Mammalian Receptor Proteins; Related Reagents and Methods

ABSTRACT

Nucleic acids encoding mammalian, e.g., primate, receptors, purified receptor proteins and fragments thereof. Antibodies, both polyclonal and monoclonal, are also provided. Methods of using the compositions for both diagnostic and therapeutic utilities are described.

This filing claims benefit of U.S. Provisional Patent Application No.60/203,426, filed May 10, 2000, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for affectingmammalian physiology, including immune system function. In particular,it provides methods to regulate development and/or the immune system.Diagnostic and therapeutic uses of these materials are also disclosed.

BACKGROUND OF THE INVENTION

Recombinant DNA technology refers generally to techniques of integratinggenetic information from a donor source into vectors for subsequentprocessing, such as through introduction into a host, whereby thetransferred genetic information is copied and/or expressed in the newenvironment. Commonly, the genetic information exists in the form ofcomplementary DNA (cDNA) derived from messenger RNA (mRNA) coding for adesired protein product. The carrier is frequently a plasmid having thecapacity to incorporate cDNA for later replication in a host and, insome cases, actually to control expression of the cDNA and therebydirect synthesis of the encoded product in the host. See, e.g.,Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, (2d ed.)vols. 1 3, CSH Press, NY.

For some time, it has been known that the mammalian immune response isbased on a series of complex cellular interactions, called the “immunenetwork”. Recent research has provided new insights into the innerworkings of this network. While it remains clear that much of the immuneresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes, and other cells, immunologistsnow generally hold the opinion that soluble proteins, known aslymphokines, cytokines, or monokines, play critical roles in controllingthese cellular interactions. Thus, there is considerable interest in theisolation, characterization, and mechanisms of action of cell modulatoryfactors, an understanding of which will lead to significant advancementsin the diagnosis and therapy of numerous medical abnormalities, e.g.,immune system disorders.

Lymphokines apparently mediate cellular activities in a variety of ways.See, e.g., Paul (ed. 1996) Fundamental Immunology 3d ed., Raven Press,New York; and Thomson (ed. 1994) The Cytokine Handbook 2d ed., AcademicPress, San Diego. They have been shown to support the proliferation,growth, and/or differentiation of pluripotential hematopoietic stemcells into vast numbers of progenitors comprising diverse cellularlineages which make up a complex immune system. Proper and balancedinteractions between the cellular components are necessary for a healthyimmune response. The different cellular lineages often respond in adifferent manner when lymphokines are administered in conjunction withother agents.

Cell lineages especially important to the immune response include twoclasses of lymphocytes: B-cells, which can produce and secreteimmunoglobulins (proteins with the capability of recognizing and bindingto foreign matter to effect its removal), and T-cells of various subsetsthat secrete lymphokines and induce or suppress the B-cells and variousother cells (including other T-cells) making up the immune network.These lymphocytes interact with many other cell types.

Research to better understand and treat various immune disorders hasbeen hampered by the general inability to maintain cells of the immunesystem in vitro. Immunologists have discovered that culturing many ofthese cells can be accomplished through the use of T-cell and other cellsupernatants, which contain various growth factors, including many ofthe lymphokines.

Various growth and regulatory factors exist which modulate morphogeneticdevelopment. Many receptors for cytokines are known. Often, there are atleast two critical subunits in the functional receptor. See, e.g.,Heinrich, et al. (1998) Biochem. J. 334:297-314; Gonda and D′Andrea(1997) Blood 89:355-369; Presky, et al. (1996) Proc. Nat'l Acad. Sci.USA 93:14002-14007; Drachman and Kaushansky (1995) Curr. Opin. Hematol.2:22-28; Theze (1994) Eur. Cytokine Netw. 5:353-368; and Lemmon andSchlessinger (1994) Trends Biochem. Sci. 19:459-463.

From the foregoing, it is evident that the discovery and development ofnew soluble proteins and their receptors, including ones similar tolymphokines, should contribute to new therapies for a wide range ofdegenerative or abnormal conditions which directly or indirectly involvedevelopment, differentiation, or function, e.g., of the immune systemand/or hematopoietic cells. In particular, the discovery andunderstanding of novel receptors for lymphokine-like molecules whichenhance or potentiate the beneficial activities of other lymphokineswould be highly advantageous. The present invention provides newreceptors for ligands exhibiting similarity to cytokine likecompositions and related compounds, and methods for their use.

SUMMARY OF THE INVENTION

The present invention is directed to novel receptors related to cytokinereceptors, e.g., primate, cytokine receptor-like molecular structures,designated DNAX Cytokine Receptor Subunits (DCRS), and their biologicalactivities. In particular, it provides description of one subunit,designated DCRS5. It includes nucleic acids coding for the polypeptidesthemselves and methods for their production and use. The nucleic acidsof the invention are characterized, in part, by their homology to clonedcomplementary DNA (cDNA) sequences enclosed herein. Additionally, theinvention provides matching of the p40/IL-B30 ligand with receptorsubunits DCRS5 and IL-12Rβ1, which pairing provides insight intoindications for use of the agonists and antagonists based upon reagentsdirected thereto.

The present invention provides a substantially pure or recombinantpolypeptide comprising at least ten contiguous amino acids of theintracellular portion of SEQ ID NO: 2. In certain embodiments, thepolypeptide: comprises at least 25 contiguous amino acids of theintracellular portion of SEQ ID NO: 2; is recombinant, comprising theintracellular portion of SEQ ID NO: 2; further comprises at least tencontiguous amino acids of the non-intracellular portion of SEQ ID NO: 2;comprises at least 25 amino acids of the extracellular portion of SEQ IDNO: 2; comprises the mature SEQ ID NO: 2; or is a substantially purenatural polypeptide. In others, the recombinant polypeptide: consists ofthe mature sequence of Table 1; is an unglycosylated polypeptide; isfrom a human; comprises at least 40 contiguous amino acids of SEQ ID NO:2; exhibits at least three nonoverlapping segments of at least fifteencontiguous amino acids of SEQ ID NO: 2; is a natural polymorphic variantof SEQ ID NO: 2; has a length at least about 30 amino acids; exhibits atleast two non-overlapping epitopes which are specific for a primateDCRS5; has a molecular weight of at least 30 kD with naturalglycosylation; is a synthetic polypeptide; is in a sterile form; is inan aqueous or buffered solution; is attached to a solid substrate; isconjugated to another chemical moiety; or is physically associated withan IL-12Rβ1 polypeptide.

Other embodiments of the invention provide: a substantially pure orrecombinant polypeptide comprising at least two distinct nonoverlappingsegments of at least six contiguous amino acids of the intracellularportion of SEQ ID NO: 2; a substantially pure or recombinant polypeptidecomprising at least twelve contiguous amino acids of the intracellularportion of SEQ ID NO: 2; or a substantially pure natural sequencepolypeptide comprising mature SEQ ID NO: 2. In particular forms, thepolypeptide comprising at least two distinct nonoverlapping segments ofat least six contiguous amino acids of the intracellular portion of SEQID NO: 2 will be where: the distinct nonoverlapping segments: includeone of at least twelve amino acids; include one of at least seven aminoacids and a second of at least nine amino acids; include a thirddistinct segment of at least six amino acids; or comprise one ofR355-L373, P378-L405, V407-D426, K428-D439, P441-V452, I454-G460,I465-T587, or N592-606; or the polypeptide further comprises at leasttwo distinct nonoverlapping segments of at least six contiguous aminoacids of the extracellular portion of SEQ ID NO: 2. Alternatively, thepolypeptide comprising at least twelve contiguous amino acids of theintracellular portion of SEQ ID NO: 2 will be one where: the at leasttwelve contiguous amino acid segment comprises one of R355-L373,P378-L405, V407-D426, K428-D439, P441-V452, I454-G460, I465-T587, orN592-606; or the polypeptide further comprises at least two distinctnonoverlapping segments of at least six contiguous amino acids of theextracellular portion of SEQ ID NO: 2. Or, the pure natural sequencepolypeptide comprising mature SEQ ID NO: 2 may further comprising apurification or detection epitope. Such polypeptides may: consist of themature sequence of Table 1; be an unglycosylated polypeptide; be from ahuman; comprise at least 40 contiguous amino acids of SEQ ID NO: 2;exhibit at least three nonoverlapping segments of at least fifteencontiguous amino acids of SEQ ID NO: 2; be a natural polymorphic variantof SEQ ID NO: 2; have a length at least about 30 amino acids; exhibit atleast two non-overlapping epitopes which are specific for a primateDCRS5; have a molecular weight of at least 30 kD with naturalglycosylation; be a synthetic polypeptide; be in a sterile form; be inan aqueous or buffered solution; be attached to a solid substrate; beconjugated to another chemical moiety; or be physically associated withan IL-12Rβ1 polypeptide.

Various other compositions are provided, e.g., comprising: asubstantially pure polypeptide combined with the IL-12Rβ1 protein; orsuch a polypeptide in a carrier, wherein the carrier is: an aqueouscompound, including water, saline, and/or buffer; and/or formulated fororal, rectal, nasal, topical, or parenteral administration.

Kits are provided comprising such a polypeptide and: a compartmentcomprising the polypeptide; a compartment comprising an IL-12Rβ1polypeptide; a compartment comprising a p40, IL-B30, or p40/IL-B30polypeptide; or instructions for use or disposal of reagents in the kit.

Antibodies and other binding compounds are provided, e.g., comprising anantigen binding site from an antibody, which specifically binds to theintracellular portion of the DCRS5, wherein: the binding compound is ina container; the polypeptide is from a human; the binding compound is anFv, Fab, or Fab2 fragment; the binding compound is conjugated to anotherchemical moiety; or the antibody: is raised against a peptide sequenceof a mature polypeptide of Table 1; is raised against a mature DCRS5; israised to a purified human DCRS5; is immunoselected; is a polyclonalantibody; binds to a denatured DCRS5; exhibits a Kd to antigen of atleast 30 μm; is attached to a solid substrate, including a bead orplastic membrane; is in a sterile composition; or is detectably labeled,including a radioactive or fluorescent label. Kits are also providedcomprising the binding compound and: a compartment comprising thebinding compound; a compartment comprising: a p40 polypeptide; an IL-B30polypeptide; a DCRS5 polypeptide; and/or an IL-12Rβ1 polypeptide; acompartment comprising an antibody which binds selectively to: a p40polypeptide; an IL-B30 polypeptide; a DCRS5 polypeptide; and/or anIL-12Rβ1 polypeptide; or instructions for use or disposal of reagents inthe kit.

Also provided are methods, e.g., of producing an antigen:antibodycomplex, comprising contacting under appropriate conditions a primateDCRS5 polypeptide with an antibody, thereby allowing the complex toform. Such method may be where: the complex is purified from othercytokine receptors; the complex is purified from other antibody; thecontacting is with a sample comprising an interferon; the contactingallows quantitative detection of the antigen; the contacting is with asample comprising the antibody; or the contacting allows quantitativedetection of the antibody. Other compositions are provided, e.g.,composition comprising: a sterile binding compound, or the bindingcompound and a carrier, wherein the carrier is: an aqueous compound,including water, saline, and/or buffer; and/or formulated for oral,rectal, nasal, topical, or parenteral administration.

The invention also provides an isolated or recombinant nucleic acidencoding the DCRS5 polypeptide, wherein the: DCRS5 is from a human; orthe nucleic acid: encodes an antigenic peptide sequence of Table 1;encodes a plurality of antigenic peptide sequences of Table 1; exhibitsidentity over at least thirteen nucleotides to a natural cDNA encodingthe segment; is an expression vector; further comprises an origin ofreplication; is from a natural source; comprises a detectable label;comprises synthetic nucleotide sequence; is less than 6 kb, preferablyless than 3 kb; is from a primate; comprises a natural full lengthcoding sequence; is a hybridization probe for a gene encoding the DCRS5;or is a PCR primer, PCR product, or mutagenesis primer. Cells comprisingthe recombinant nucleic acid are provided, including where the cell is:a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell;an insect cell; a mammalian cell; a mouse cell; a primate cell; or ahuman cell.

Kit embodiments include those comprising the nucleic acid and: acompartment comprising the nucleic acid; a compartment comprising anucleic acid encoding: a p40 polypeptide; an IL-B30 polypeptide; a DCRS5polypeptide; and/or an IL-12Rβ1 polypeptide; a compartment comprising: ap40 polypeptide; an IL-B30 polypeptide; a DCRS5 polypeptide; and/or anIL-12Rβ1 polypeptide; a compartment comprising an antibody whichselectively binds to: a p40 polypeptide; an IL-B30 polypeptide; a DCRS5polypeptide; and/or an IL-12Rβ1 polypeptide; or instructions for use ordisposal of reagents in the kit.

Other nucleic acid embodiments include those which: hybridize under washconditions of 30 minutes at 30° C. and less than 2M salt to the portionof SEQ ID NO: 1 encoding the intracellular portion; or exhibit identityover a stretch of at least about 30 nucleotides to the intracellularportion of a primate DCRS5. Preferably, such nucleic acid will be onewherein: the wash conditions are at 45° C. and/or 500 mM salt; or 55° C.and/or 150 mM salt; or the stretch is at least 55 or 75 nucleotides.

Therapeutic uses include methods of modulating physiology or developmentof a cell comprising contacting the cell with: an antagonist ofp40/IL-B30 which is a complex comprising: the extracellular portion of aprimate DCRS5 and/or the extracellular portion of a primate IL-12Rβ1; anantagonist of p40/IL-B30 which is an antibody which binds a complexcomprising: primate DCRS5 and/or primate IL-12Rβ1; an antagonist ofp40/IL-B30 which is an antibody which binds to DCRS5; an antagonist ofp40/IL-B30 which is an antibody to IL-12Rβ1; an antagonist of p40/IL-B30which is an antisense nucleic acid to DCRS5 or IL-12Rβ1; or an agonistof p40/IL-B30 which is an antibody which binds a complex comprising:primate DCRS5 and/or primate IL-12Rβ1. In one type of method, thecontacting is with an antagonist, and the contacting is in combinationwith an antagonist to Il-12, IL-18, TNF, and/or IFNγ; or the cell isfrom a host which: exhibits signs of symptoms of a chronic TH1 mediateddisease; exhibits symptoms or signs of multiple sclerosis, rheumatoidarthritis, osteoarthritis, inflammatory bowel disease, diabetes,psoriasis, or sepsis; or receives an allogeneic transplant. Conversely,the method may be contacting with an agonist, and: the contacting is incombination with IL-12, IL-18, TNF, or IFNγ; or the cell is from a hostwhich: exhibits signs or symptoms of a chronic Th2 response; suffersfrom a tumor, viral, or fungal growth; receives a vaccine; or suffersfrom an allergic response.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Outline I. General II.Activities

III. Nucleic acids

A. encoding fragments, sequence, probes

B. mutations, chimeras, fusions

C. making nucleic acids

D. vectors, cells comprising

IV. Proteins, Peptides

A. fragments, sequence, immunogens, antigens

B. muteins

C. agonists/antagonists, functional equivalents

D. making proteins

V. Making nucleic acids, proteins

A. synthetic

B. recombinant

C. natural sources

VI. Antibodies

A. polyclonals

B. monoclonal

C. fragments; Kd

D. anti-idiotypic antibodies

E. hybridoma cell lines

VII. Kits, Diagnosis, and Quantitation

A. ELISA

B. assay mRNA encoding

C. qualitative/quantitative

D. kits

VIII. Therapeutic compositions, methods

A. combination compositions

B. unit dose

C. administration

IX. Screening I. General

The present invention provides the amino acid sequence and DNA sequenceof mammalian, herein primate, cytokine receptor-like subunit molecules,this one designated DNAX Cytokine Receptor Subunit 5 (DCRS5) havingparticular defined properties, both structural and biological. VariouscDNAs encoding these molecules were obtained from primate, e.g., human,cDNA sequence libraries. Other primate or other mammalian counterpartswould also be desired.

Additionally, the invention provides matching of the p40/IL-B30 ligandwith receptor subunits DCRS5 and IL-12Rβ1, which pairing providesinsight into indications for use of the agonists and antagonists basedupon reagents directed thereto.

Some of the standard methods applicable are described or referenced,e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, etal. (1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1 3,CSH Press, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and periodic supplements)Current Protocols in Molecular Biology, Greene/Wiley, New York; each ofwhich is incorporated herein by reference.

Nucleotide (SEQ ID NO: 1) and corresponding amino acid sequence (SEQ IDNO: 2) of a primate, e.g., human, DCRS5 coding segment is shown inTable 1. The predicted signal sequence is indicated, but may depend oncell type, or may be a few residues in either direction. Potential Nglycosylation sites are at Asparagine residues 6, 24, 58, 118, 157, 209,and 250. Disulfide linkages are likely to be found between cysteineresidues at positions 29 and 78; and a conserved C_CXW motif is found atpositions 110/121/123. The tryptophan at 219; and the WxxWS motif from281-285 are notable. The segment from about 1-101 is an Ig domain; fromabout 102-195 is a cytokine binding domain 1; from about 196-297 is acytokine binding domain 2; from about 298-330 is a linker; from about329-354 is a transmembrane segment; and from about 356-606 is anintracellular domain. Intracellular features include putative SH2binding sites at Y374-1377, Y461-Q464, and Y588-Q591; and potentiallyimportant tyrosine residues at 406, 427, 440, and 453. These sites andboundaries are notable.

The ORF contains a putative signal sequence which is predicted to becleaved at . . . CHG/GIT . . . as shown above. A predicted extracellulardomain of 328 amino acids is followed by a putative transmembranesegment, and finally a cytoplasmic domain of about 252 amino acids. Theligand-binding functions are predicted to reside in the extracellulardomain.

The reverse translation nucleic acid sequence is provided in Table 2.

TABLE 1 Nucleotide and polypeptide sequences of DNAX Cytokine ReceptorSubunit like embodiments (DCRS5). Primate, e.g., human embodiment (seeSEQ ID NO: 1 and 2). Predicted signal sequence indicated, but may varyby a few positions and depending upon cell type. Identified positions ofvariation are at nucleotides 127 and 563; which are paired G and G(translating to the combination of Q and G) or T and A (translating to Hand R). gtggtacggg aattccattg tgttgggcag ccaacaaggg tggcagcctggctctgaagt   60 ggaattatgt gcttcaaaca ggttgaaaga gggaaacagt cttttcctgcttccagac  118 atg aat cak gtc act att caa tgg gat gca gta ata gcc ctttac ata  166 Met Asn Xaa Val Thr Ile Gln Trp Asp Ala Val Ile Ala Leu TyrIle             −20                 −15                 −10 ctc ttc agctgg tgt cat gga gga att aca aat ata aac tgc tct ggc  214 Leu Phe Ser TrpCys His Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly         −5              −1   1               5 cac atc tgg gta gaa ccagcc aca att ttt aag atg ggt atg aat atc  262 His Ile Trp Val Glu Pro AlaThr Ile Phe Lys Met Gly Met Asn Ile 10                  15                  20                  25 tct atatat tgc caa gca gca att aag aac tgc caa cca agg aaa ctt  310 Ser Ile TyrCys Gln Ala Ala Ile Lys Asn Cys Gln Pro Arg Lys Leu                 30                  35                  40 cat ttt tataaa aat ggc atc aaa gaa aga ttt caa atc aca agg att  358 His Phe Tyr LysAsn Gly Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile             45                  50                  55 aat aaa aca acagct cgg ctt tgg tat aaa aac ttt ctg gaa cca cat  406 Asn Lys Thr Thr AlaArg Leu Trp Tyr Lys Asn Phe Leu Glu Pro His         60                  65                  70 gct tct atg tac tgcact gct gaa tgt ccc aaa cat ttt caa gag aca  454 Ala Ser Met Tyr Cys ThrAla Glu Cys Pro Lys His Phe Gln Glu Thr     75                  80                  85 ctg ata tgt gga aaa gacatt tct tct gga tat ccg cca gat att cct  502 Leu Ile Cys Gly Lys Asp IleSer Ser Gly Tyr Pro Pro Asp Ile Pro 90                  95                 100                 105 gat gaagta acc tgt gtc att tat gaa tat tca ggc aac atg act tgc  550 Asp Glu ValThr Cys Val Ile Tyr Glu Tyr Ser Gly Asn Met Thr Cys                110                 115                 120 acc tgg aatgct rgg aag ctc acc tac ata gac aca aaa tac gtg gta  598 Thr Trp Asn AlaXaa Lys Leu Thr Tyr Ile Asp Thr Lys Tyr Val Val            125                 130                 135 cat gtg aag agttta gag aca gaa gaa gag caa cag tat ctc acc tca  646 His Val Lys Ser LeuGlu Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser        140                 145                 150 agc tat att aac atctcc act gat tca tta caa ggt ggc aag aag tac  694 Ser Tyr Ile Asn Ile SerThr Asp Ser Leu Gln Gly Gly Lys Lys Tyr    155                 160                 165 ttg gtt tgg gtc caa gcagca aac gca cta ggc atg gaa gag tca aaa  742 Leu Val Trp Val Gln Ala AlaAsn Ala Leu Gly Met Glu Glu Ser Lys170                 175                 180                 185 caa ctgcaa att cac ctg gat gat ata gtg ata cct tct gca gcc gtc  790 Gln Leu GlnIle His Leu Asp Asp Ile Val Ile Pro Ser Ala Ala Val                190                 195                 200 att tcc agggct gag act ata aat gct aca gtg ccc aag acc ata att  838 Ile Ser Arg AlaGlu Thr Ile Asn Ala Thr Val Pro Lys Thr Ile Ile            205                 210                 215 tat tgg gat agtcaa aca aca att gaa aag gtt tcc tgt gaa atg aga  886 Tyr Trp Asp Ser GlnThr Thr Ile Glu Lys Val Ser Cys Glu Met Arg        220                 225                 230 tac aag gct aca acaaac caa act tgg aat gtt aaa gaa ttt gac acc  934 Tyr Lys Ala Thr Thr AsnGln Thr Trp Asn Val Lys Glu Phe Asp Thr    235                 240                 245 aat ttt aca tat gtg caacag tca gaa ttc tac ttg gag cca aac att  982 Asn Phe Thr Tyr Val Gln GlnSer Glu Phe Tyr Leu Glu Pro Asn Ile250                 255                 260                 265 aag tacgta ttt caa gtg aga tgt caa gaa aca ggc aaa agg tac tgg 1030 Lys Tyr ValPhe Gln Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp                270                 275                 280 cag cct tggagt tca ccg ttt ttt cat aaa aca cct gaa aca gtt ccc 1078 Gln Pro Trp SerSer Pro Phe Phe His Lys Thr Pro Glu Thr Val Pro            285                 290                 295 cag gtc aca tcaaaa gca ttc caa cat gac aca tgg aat tct ggg cta 1126 Gln Val Thr Ser LysAla Phe Gln His Asp Thr Trp Asn Ser Gly Leu        300                 305                 310 aca gtt gct tcc atctct aca ggg cac ctt act tct gac aac aga gga 1174 Thr Val Ala Ser Ile SerThr Gly His Leu Thr Ser Asp Asn Arg Gly    315                 320                 325 gac att gga ctt tta ttggga atg atc gtc ttt gct gtt atg ttg tca 1222 Asp Ile Gly Leu Leu Leu GlyMet Ile Val Phe Ala Val Met Leu Ser330                 335                 340                 345 att ctttct ttg att ggg ata ttt aac aga tca ttc cga act ggg att 1270 Ile Leu SerLeu Ile Gly Ile Phe Asn Arg Ser Phe Arg Thr Gly Ile                350                 355                 360 aaa aga aggatc tta ttg tta ata cca aag tgg ctt tat gaa gat att 1318 Lys Arg Arg IleLeu Leu Leu Ile Pro Lys Trp Leu Tyr Glu Asp Ile            365                 370                 375 cct aat atg aaaaac agc aat gtt gtg aaa atg cta cag gaa aat agt 1366 Pro Asn Met Lys AsnSer Asn Val Val Lys Met Leu Gln Glu Asn Ser        380                 385                 390 gaa ctt atg aat aataat tcc agt gag cag gtc cta tat gtt gat ccc 1414 Glu Leu Met Asn Asn AsnSer Ser Glu Gln Val Leu Tyr Val Asp Pro    395                 400                 405 atg att aca gag ata aaagaa atc ttc atc cca gaa cac aag cct aca 1462 Met Ile Thr Glu Ile Lys GluIle Phe Ile Pro Glu His Lys Pro Thr410                 415                 420                 425 gac tacaag aag gag aat aca gga ccc ctg gag aca aga gac tac ccg 1510 Asp Tyr LysLys Glu Asn Thr Gly Pro Leu Glu Thr Arg Asp Tyr Pro                430                 435                 440 caa aac tcgcta ttc gac aat act aca gtt gta tat att cct gat ctc 1558 Gln Asn Ser LeuPhe Asp Asn Thr Thr Val Val Tyr Ile Pro Asp Leu            445                 450                 455 aac act gga tataaa ccc caa att tca aat ttt ctg cct gag gga agc 1606 Asn Thr Gly Tyr LysPro Gln Ile Ser Asn Phe Leu Pro Glu Gly Ser        460                 465                 470 cat ctc agc aat aataat gaa att act tcc tta aca ctt aaa cca cca 1654 His Leu Ser Asn Asn AsnGlu Ile Thr Ser Leu Thr Leu Lys Pro Pro    475                 480                 485 gtt gat tcc tta gac tcagga aat aat ccc agg tta caa aag cat cct 1702 Val Asp Ser Leu Asp Ser GlyAsn Asn Pro Arg Leu Gln Lys His Pro490                 495                 500                 505 aat tttgct ttt tct gtt tca agt gtg aat tca cta agc aac aca ata 1750 Asn Phe AlaPhe Ser Val Ser Ser Val Asn Ser Leu Ser Asn Thr Ile                510                 515                 520 ttt ctt ggagaa tta agc ctc ata tta aat caa gga gaa tgc agt tct 1798 Phe Leu Gly GluLeu Ser Leu Ile Leu Asn Gln Gly Glu Cys Ser Ser            525                 530                 535 cct gac ata caaaac tca gta gag gag gaa acc acc atg ctt ttg gaa 1846 Pro Asp Ile Gln AsnSer Val Glu Glu Glu Thr Thr Met Leu Leu Glu        540                 545                 550 aat gat tca ccc agtgaa act att cca gaa cag acc ctg ctt cct gat 1894 Asn Asp Ser Pro Ser GluThr Ile Pro Glu Gln Thr Leu Leu Pro Asp    555                 560                 565 gaa ttt gtc tcc tgt ttgggg atc gtg aat gag gag ttg cca tct att 1942 Glu Phe Val Ser Cys Leu GlyIle Val Asn Glu Glu Leu Pro Ser Ile570                 575                 580                 585 aat acttat ttt cca caa aat att ttg gaa agc cac ttc aat agg att 1990 Asn Thr TyrPhe Pro Gln Asn Ile Leu Glu Ser His Phe Asn Arg Ile                590                 595                 600 tca ctc ttggaa aag tagagctgtg tggtcaaaat caatatgaga aagctgcctt 2045 Ser Leu Leu GluLys             605 gcaatctgaa cttgggtttt ccctgcaata gaaattgaattctgcctctt tttgaaaaaa 2105 atgtattcac atacaaatct tcacatggac acatgttttcatttcccttg gataaatacc 2165 taggtagggg attgctgggc catatgataa gcatatgtttcagttctacc aatcttgttt 2225 ccagagtagt gacatttctg tgctcctacc atcaccatgtaagaattccc gggagctcca 2285 tgccttttta attttagcca ttcttctgcc tmatttcttaaaattagaga attaaggtcc 2345 cgaaggtgga acatgcttca tggtcacaca tacaggcacaaaaacagcat tatgtggacg 2405 cctcatgtat tttttataga gtcaactatt tcctctttattttccctcat tgaaagatgc 2465 aaaacagctc tctattgtgt acagaaaggg taaataatgcaaaatacctg gtagtaaaat 2525 aaatgctgaa aattttcctt taaaatagaa tcattaggccaggcgtggtg gctcatgctt 2585 gtaatcccag cactttggta ggctgaggtr ggtggatcacctgaggtcag gagttcgagt 2645 ccagcctggc caatatgctg aaaccctgtc tctactaaaattacaaaaat tagccggcca 2705 tggtggcagg tgcttgtaat cccagctact tgggaggctgaggcaggaga atcacttgaa 2765 ccaggaaggc agaggttgca ctgagctgag attgtgccactgcactccag cctgggcaac 2825 aagagcaaaa ctctgtctgg aaaaaaaaaa aaaa 2859MN(Q/H)VTIQWDAVIALYILFSWCHGGITNINCSGHIWVEPATIFKMGMNISIYCQAAIKNCQPRKLHFYKNGIKERFQITRINKTTARLWYKNFLEPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMTCTWNA(G/R)KLTYIDTKYVVHVKSLETEEEQQYLTSSYINISTDSLQGGKKYLVWVQAANALGMEESKQLQIHLDDIVIPSAAVISRAETINATVPKTIIYWDSQTTIEKVSCEMRYKATTNQTWNVKEFDTNFTYVQQSEFYLEPNIKYVFQVRCQETGKRYWQPWSSPFFHKTPETVPQVTSKAFQHDTWNSGLTVASISTGHLTSDNRGDIGLLLGMIVFAVMLSILSLIGIFNRSFRTGIKRRILLLIPKWL

PNMKNSNVVKMLQENSELMNNNSSEQVLYVDPMITEIKEIFIPEHKPTDYKKENTGPLETRDYPQNSLFDNTTVVYIPDLNT G

KPQISNFLPEGSHLSNNNEITSLTLKPPVDSLDSGNNPRLQKHPNFAFSVSSVNSLSNTIFLGELSLILNQGECSSPDIQNSVEEETTMLLENDSPSETIPEQTLLPDEFVSCLGIVNEELPSINT

FPQNILESHFN RISLLEK

TABLE 2 Reverse Translation of primate, e.g., human, DCRS5 (SEQ ID NO:3):ATGAAYCAYGTNACNATHCARTGGGAYGCNGTNATHGCNYTNTAYATHYTNTTYWSNTGGTGYCAYGGNGGNATHACNAAYATHAAYTGYWSNGGNCAYATHTGGGTNGARCCNGCNACNATHTTYAARATGGGNATGAAYATHWSNATHTAYTGYCARGCNGCNATHAARAAYTGYCARCCNMGNAARYTNCAYTTYTAYAARAAYGGNATHAARGARMGNTTYCARATHACNMGNATHAAYAARACNACNGCNMGNYTNTGGTAYAARAAYTTYYTNGARCCNCAYGCNWSNATGTAYTGYACNGCNGARTGYCCNAARCAYTTYCARGARACNYTNATHTGYGGNAARGAYATHWSNWSNGGNTAYCCNCCNGAYATHCCNGAYGARGTNACNTGYGTNATHTAYGARTAYWSNGGNAAYATGACNTGYACNTGGAAYGCNMGNAARYTNACNTAYATHGAYACNAARTAYGTNGTNCAYGTNAARWSNYTNGARACNGARGARGARCARCARTAYYTNACNWSNWSNTAYATHAAYATHWSNACNGAYWSNYTNCARGGNGGNAARAARTAYYTNGTNTGGGTNCARGCNGCNAAYGCNYTNGGNATGGARGARWSNAARCARYTNCARATHCAYYTNGAYGAYATHGTNATHCCNWSNGCNGCNGTNATHWSNMGNGCNGARACNATHAAYGCNACNGTNCCNAARACNATHATHTAYTGGGAYWSNCARACNACNATHGARAARGTNWSNTGYGARATGMGNTAYAARGCNACNACNAAYCARACNTGGAAYGTNAARGARTTYGAYACNAAYTTYACNTAYGTNCARCARWSNGARTTYTAYYTNGARCCNAAYATHAARTAYGTNTTYCARGTNMGNTGYCARGARACNGGNAARMGNTAYTGGCARCCNTGGWSNWSNCCNTTYTTYCAYAARACNCCNGARACNGTNCCNCARGTNACNWSNAARGCNTTYCARCAYGAYACNTGGAAYWSNGGNYTNACNGTNGCNWSNATHWSNACNGGNCAYYTNACNWSNGAYAAYMGNGGNGAYATHGGNYTNYTNYTNGGNATGATHGTNTTYGCNGTNATGYTNWSNATHYTNWSNYTNATHGGNATHTTYAAYMGNWSNTTYMGNACNGGNATHAARMGNMGNATHYTNYTNYTNATHCCNAARTGGYTNTAYGARGAYATHCCNAAYATGAARAAYWSNAAYGTNGTNAARATGYTNCARGARAAYWSNGARYTNATGAAYAAYAAYWSNWSNGARCARGTNYTNTAYGTNGAYCCNATGATHACNGARATHAARGARATHTTYATHCCNGARCAYAARCCNACNGAYTAYAARAARGARAAYACNGGNCCNYTNGARACNMGNGAYTAYCCNCARAAYWSNYTNTTYGAYAAYACNACNGTNGTNTAYATHCCNGAYYTNAAYACNGGNTAYAARCCNCARATHWSNAAYTTYYTNCCNGARGGNWSNCAYYTNWSNAAYAAYAAYGARATHACNWSNYTNACNYTNAARCCNCCNGTNGAYWSNYTNGAYWSNGGNAAYAAYCCNMGNYTNCARAARCAYCCNAAYTTYGCNTTYWSNGTNWSNWSNGTNAAYWSNYTNWSNAAYACNATHTTYYTNGGNGARYTNWSNYTNATHYTNAAYCARGGNGARTGYWSNWSNCCNGAYATHCARAAYWSNGTNGARGARGARACNACNATGYTNYTNGARAAYGAYWSNCCNWSNGARACNATHCCNGARCARACNYTNYTNCCNGAYGARTTYGTNWSNTGYYTNGGNATHGTNAAYGARGARYTNCCNWSNATHAAYACNTAYTTYCCNCARAAYATHYTNGARWSNCAYTTYAAYMGNATHWSNYTNYTNGARAAR

TABLE 3 Alignment of various cytokine receptor subunits. Human IL-6receptor protein gp130 is SEQ ID NO: 4 (GenBank M57230); human IL-12receptor beta2 subunit is SEQ ID NO: 5 (GenBank U64198). huIL-12R 2   1  MAHTFRGCSLAFMFIITWLLIKAKIDACKRGDVTVKPSHVILLGSTVN  48 hugp130   1  MLTLQTWVVQALFIFLTTESTGELLDPCG---YISPESPVVQLHSNFT  45 huDCRS5   1MNHVTIQWDAVIALYILFSWCHGGITNINCS-GHIWVEPATIFKMGMNIS  49            *     .          *         . .  .  . huIL-12R 2  49ITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLG---  95 hugp130  46AVCVLKEKCMDYFHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIA  95 huDCRS5  50IYCQAAIKN--CQP---RKLHFYKNGIKER-FQITRINKTTARLWYKNFL  93  *    .           .            .   .*   . . huIL-12R 2  96--TTLFVCKLACINSD-EIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVA 142 hugp130  96SLNIQLTCNILTFGQL-EQNVYGITIISGLPPEKPKNLSCIVN-EGKKMR 143 huDCRS5  94EPHASMYCTAECPKHFQETLICGKDISSGYPPDIPDEVTCVIYEYSGNMT 143       *   .     *  . *  *  *  *. *  ..*.       . huIL-12R 2 143CTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESP 192 hugp130 144CEWDGGRETHLETNFTLKS--EWATHKFADCKAKRDTPTSCTVDYS-TVY 190 huDCRS5 144CTWNARKLTYIDTKYVVHVKSLETEEEQQYLTSSYINISTDSLQGG---- 189 * *   . * . * ...                        . huIL-12R 2 193ESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRC 242 hugp130 191FVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSIL 240 huDCRS5 190-KKYLVWVQAANALGMEESKQLQIHLDDIVIPSAAVISRAETINATVPKT 238        * **.**   *          * * huIL-12R 2 243TLYWRD----EGLVLLNRLRYRPSNSRLWNMVN---VTKAKGRHDLLDLK 285 hugp130 241KLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLK 290 huDCRS5 239IIYWDS--QTTIEKVSCEMRYKATTNQTWNVKEFD-TNFTYVQQSEFYLE 285 .*          .   ..*.      *          .        * huIL-12R 2 286PFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPTGMLDVWYMKRHID 335 hugp130 291PFTEYVFRIRCMKEDGKGYWSDWSEEASGITYEDRPSKAPSFWYKIDPSH 340 huDCRS5 286PNIKYVFQVRCQ-ETGKRYWQPWSSPFFHKTPETVP-------------- 320 *   * *...     *  *  **      * *  * huIL-12R 2 336YS-RQQISLFWKNLSVSEARGKILHYQVTLQELTGGKAMTQNITGHTSWT 384 hugp130 341TQGYRTVQLVWKTLPPFEANGKILDYEVT---LTRWKSHLQNYTVNATKL 387 huDCRS5 321-----QVTSKAFQHDTWNSGLTVASISTG------HLTSDN--RGDIGLL 357      .           .   .              .  . huIL-12R 2 385TVIPRTGNWAVAVSAANSKGSSLPTRINIMNLCEAGLLAPRQVSANSEGM 434 hugp130 388TVNLTNDRYLATLTVRNLVGKSDAAVLTIP-ACDFQATHPVMDLKAFPKD 436 huDCRS5 358LGMIVFAVMLSILSLIGIFNRSFRTGIKRR-------------------- 387            ..  .  . *  . . huIL-12R 2 435DNILVTWQPPRKDPSAVQEYVVEWRELHPG-GDTQVPLNWLRSRPYNVSA 483 hugp130 437NMLWVEWTTPRE---SVKKYILEWCVLS---DKAPCITDWQQEDGTVHRT 480 huDCRS5 388----------------ILLLIPKWLYEDIPNMKNSNVVKMLQEN----SE 417                .   .  *                 . huIL-12R 2 484LISENIKSYICYEIRVYALSGDQ-GGCSSILGNSKHKAPLSGPHINAITE 532 hugp130 481YLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKGPTVRTKKV 530 huDCRS5 418LMNNNSSE--------QVLYVDP-----MITEIKEIFIPEHKPTDYKKE- 453 .  *             .  *       *        *   * huIL-12R 2 533EKGSILISWNSIPVQEQMGCLLHYRIYWKERDSNSQPQLCEIPYRVSQNS 582 hugp130 531GKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGN----ETAVNVDSSHTE 576 huDCRS5 454--NTGPLETRDYP---QNSLFDNTTVVYIPDLNTG------YKPQISN-- 490  .   .     *   *     .  .       .            * huIL-12R 2 583HPINSLQPRVTYVLWMTALTAAGESSHGNEREFCLQGKAN-WMAFVAPSI 631 hugp130 577YTLSSLTSDTLYMVRMAAYTDEG-GKDGPEFTFTTPKFAQGEIEAIVVPV 625 huDCRS5 491------------------FLPEG--------------------------- 495                      * huIL-12R 2 632CIAIIMVGIFSTHYFQQKVFVLLAALRP-----------QWCSREIPDPA 670 hugp130 626CLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHN 675 huDCRS5 496-----------SHLSNNN-EITSLTLKP--------------PVDSLDSG 519                .   .    . * huIL-12R 2 671NSTCAKKYPIAEEKTQLPLDRLLID-WPTPEDPEPLVIS--EVLHQVTPV 717 hugp130 676FNSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHS 725 huDCRS5 520NNPRLQKHPN-FAFSVSSVNSLSNT-------------I---FLGELSLI 552     .         .                           . huIL-12R 2 718FRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLY 767 hugp130 726SGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQ 775 huDCRS5 553LNQGECS---S--PDIQNSVEEETTMLLENDSP----------------- 580     .*        *      * huIL-12R 2 768KVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSN---IDDLPSHEAP 814 hugp130 776VFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQHESS 825 huDCRS5 581--SETIPEQTLLPDEFVSCLGIVNEELPSINTYFPQN---ILESHFNR-- 623    .                     .       * .         . huIL-12R 2 815LADSLEELEPQHISLS-----VFPSSSLHPLTFSCG-------------- 845 hugp130 826PDISHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAAD 875 huDCRS5 624--ISLLEK 629    *  * 629 huIL-12R 2 846 ----------DKLTLDQLKMRCDSLML 862hugp130 876 AFGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMPQ 918 huDCRS5 630629

The closest relatives of the extracellular domain of “IL-30R” are theIL-6 signal transducer gp130 and IL-12Rβ2. Somewhat less close relativesare GCSF receptor, leptin receptor, leukemia inhibitory factor receptor,and CNTF receptor. Thus “IL-30R” is a member of the class I branch ofthe cytokine receptor superfamily and is closely related to theIL-6R/IL-12R family.

Table 3 shows comparison of the available sequences of primate receptorsubunits with the primate, e.g., human DCRS5 (IL-30R). The DCRS5 showssimilarity to the IL-6 receptor subunit gp130 (e.g., IL-6R subunit) andthe IL-12Rβ2 subunit. The DCRS5 exhibits structural features of a betasubunit, but the actual sequence of protein interactions and signalingremains unresolved.

As used herein, the term DCRS5 shall be used to describe a proteincomprising the amino acid sequence shown in Table 1. In many cases, asubstantial fragment thereof will be functionally or structurallyequivalent, including, e.g., additional extracellular segments. Theinvention also includes a protein variation of the respective DCRS5allele whose sequence is provided, e.g., a mutein or other construct.Typically, such variants will exhibit less than about 10% sequencedifferences with the target region, and thus will often have between 1-and 11-fold substitutions, e.g., 2-, 3-, 5, 7-fold, and others. It alsoencompasses allelic and other variants, e.g., natural polymorphisms, ofthe protein described. Typically, it will bind to its correspondingbiological ligand, perhaps in a dimerized state with an alpha receptorsubunit, with high affinity, e.g., at least about 100 nM, usually betterthan about 30 nM, preferably better than about 10 nM, and morepreferably at better than about 3 nM. The term shall also be used hereinto refer to related naturally occurring forms, e.g., alleles,polymorphic variants, and metabolic variants of the mammalian protein.Preferred forms of the receptor complexes will bind the appropriateligand with an affinity and selectivity appropriate for aligand-receptor interaction.

This invention also encompasses combinations of proteins or peptideshaving substantial amino acid sequence identity with the amino acidsequence in Table 1. It will include sequence variants with relativelyfew substitutions, e.g., preferably fewer than about 3-5.

A substantial polypeptide “fragment”, or “segment”, is a stretch ofamino acid residues of at least about 8 amino acids, generally at least10 amino acids, more generally at least 12 amino acids, often at least14 amino acids, more often at least 16 amino acids, typically at least18 amino acids, more typically at least 20 amino acids, usually at least22 amino acids, more usually at least 24 amino acids, preferably atleast 26 amino acids, more preferably at least 28 amino acids, and, inparticularly preferred embodiments, at least about 30 or more aminoacids. Sequences of segments of different proteins can be compared toone another over appropriate length stretches. In many situations,fragments may exhibit functional properties of the intact subunits,e.g., the extracellular domain of the transmembrane receptor may retainthe ligand binding features, and may be used to prepare a solublereceptor-like complex.

Amino acid sequence homology, or sequence identity, is determined byoptimizing residue matches. In some comparisons, gaps may be introduces,as required. See, e.g., Needleham, et al., (1970) J. Mol. Biol.48:443-453; Sankoff, et al., (1983) chapter one in Time Warps, StringEdits, and Macromolecules: The Theory and Practice of SequenceComparison, Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group (GCG), Madison, Wis.; each of which isincorporated herein by reference. This changes when consideringconservative substitutions as matches. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. Homologous amino acid sequences are intended toinclude natural allelic and interspecies variations in the cytokinesequence. Typical homologous proteins or peptides will have from 50 100%homology (if gaps can be introduced), to 60 100% homology (ifconservative substitutions are included) with an amino acid sequencesegment of Table 1. Homology measures will be at least about 70%,generally at least 76%, more generally at least 81%, often at least 85%,more often at least 88%, typically at least 90%, more typically at least92%, usually at least 94%, more usually at least 95%, preferably atleast 96%, and more preferably at least 97%, and in particularlypreferred embodiments, at least 98% or more. The degree of homology willvary with the length of the compared segments. Homologous proteins orpeptides, such as the allelic variants, will share most biologicalactivities with the embodiments described in Table 1, particularly theintracellular portion.

As used herein, the term “biological activity” is used to describe,without limitation, effects on signaling, inflammatory responses, innateimmunity, and/or morphogenic development by cytokine-like ligands. Forexample, these receptors should mediate phosphatase or phosphorylaseactivities, which activities are easily measured by standard procedures.See, e.g., Hardie, et al. (eds. 1995) The Protein Kinase FactBook vols.I and II, Academic Press, San Diego, Calif.; Hanks, et al. (1991) Meth.Enzymol. 200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin (1990)Cell 61:743-752; Pines, et al. (1991) Cold Spring Harbor Symp. Quant.Biol. 56:449-463; and Parker, et al. (1993) Nature 363:736-738. Thereceptors, or portions thereof, may be useful as phosphate labelingenzymes to label general or specific substrates. The subunits may alsobe functional immunogens to elicit recognizing antibodies, or antigenscapable of binding antibodies.

The terms ligand, agonist, antagonist, and analog of, e.g., a DCRS5petition features of ligand-receptor interactions, e.g., where thereceptor is a natural receptor or an antibody. The cellular responseslikely are typically mediated through receptor tyrosine kinase pathways.

Also, a ligand is a molecule which serves either as a natural ligand towhich said receptor, or an analog thereof, binds, or a molecule which isa functional analog of the natural ligand. The functional analog may bea ligand with structural modifications, or may be a wholly unrelatedmolecule which has a molecular shape which interacts with theappropriate ligand binding determinants. The ligands may serve asagonists or antagonists, see, e.g., Goodman, et al. (eds. 1990) Goodman& Gilman's: The Pharmacological Bases of Therapeutics, Pergamon Press,New York.

Rational drug design may also be based upon structural studies of themolecular shapes of a receptor or antibody and other effectors orligands. See, e.g., Herz, et al. (1997) J. Recept. Signal Transduct.Res. 17:671-776; and Chaiken, et al. (1996) Trends Biotechnol.14:369-375. Effectors may be other proteins which mediate otherfunctions in response to ligand binding, or other proteins whichnormally interact with the receptor. One means for determining whichsites interact with specific other proteins is a physical structuredetermination, e.g., x-ray crystallography or 2 dimensional NMRtechniques. These will provide guidance as to which amino acid residuesform molecular contact regions. For a detailed description of proteinstructural determination, see, e.g., Blundell and Johnson (1976) ProteinCrystallography, Academic Press, New York, which is hereby incorporatedherein by reference.

II. Activities

The cytokine receptor-like proteins will have a number of differentbiological activities, e.g., intracellular signaling, e.g., via STAT4,modulating cell proliferation, or in phosphate metabolism, being addedto or removed from specific substrates, typically proteins. Such willgenerally result in modulation of an inflammatory function, other innateimmunity response, or a morphological effect. The subunit will probablyhave a specific low affinity binding to the ligand.

The DCRS5 has the characteristic motifs of a receptor signaling throughthe JAK pathway. See, e.g., Ihle, et al. (1997) Stem Cells 15 (suppl.1):105-111; Silvennoinen, et al. (1997) APMIS 105:497-509; Levy (1997)Cytokine Growth Factor Review 8:81-90; Winston and Hunter (1996) CurrentBiol. 6:668-671; Barrett (1996) Baillieres Clin. Gastroenterol. 10:1-15;and Briscoe, et al. (1996) Philos. Trans. R. Soc. Lond. B. Biol. Sci.351:167-171. Of particular interest are the SH2 binding motifs describedabove.

The biological activities of the cytokine receptor subunits will berelated to addition or removal of phosphate moieties to substrates,typically in a specific manner, but occasionally in a non specificmanner. Substrates may be identified, or conditions for enzymaticactivity may be assayed by standard methods, e.g., as described inHardie, et al. (eds. 1995) The Protein Kinase FactBook vols. I and II,Academic Press, San Diego, Calif.; Hanks, et al. (1991) Meth. Enzymol.200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin (1990) Cell61:743-752; Pines, et al. (1991) Cold Spring Harbor Symp. Quant. Biol.56:449-463; and Parker, et al. (1993) Nature 363:736-738.

The receptor subunits may combine to form functional complexes, e.g.,which may be useful for binding ligand or preparing antibodies. Thesewill have substantial diagnostic uses, including detection orquantitation. The functional linkage of the receptor with the p40/IL-B30ligand provides important insights into the clinical indications thatthe receptor will be useful for. Thus, antagonists and agonists willhave predicted functional effects.

III. Nucleic Acids

This invention contemplates use of isolated nucleic acid or fragments,e.g., which encode these or closely related proteins, or fragmentsthereof, e.g., to encode a corresponding polypeptide, preferably onewhich is biologically active. In addition, this invention coversisolated or recombinant DNAs which encode combinations of such proteinsor polypeptides having characteristic sequences, e.g., of the DCRS5salone or in combination with others such as an IL-12Rβ1 (see Showe, etal. (1996) Ann. N.Y. Acad. Sci. 795:413-425; Gately, et al. (1998) Ann.Rev. Immunol. 16:495-521; GenBank U03187, NM_(—)005535) subunit.Typically, the nucleic acid is capable of hybridizing, under appropriateconditions, with a nucleic acid sequence segment shown in Table 1, butpreferably not with a corresponding segment of other receptors describedin Table 3. Said biologically active protein or polypeptide can be afull length protein, or fragment, and will typically have a segment ofamino acid sequence highly homologous, e.g., exhibiting significantstretches of identity, to one shown in Table 1. Further, this inventioncovers the use of isolated or recombinant nucleic acid, or fragmentsthereof, which encode proteins having fragments which are equivalent tothe DCRS5 proteins, e.g., intracellular portions. The isolated nucleicacids can have the respective regulatory sequences in the 5′ and 3′flanks, e.g., promoters, enhancers, poly-A addition signals, and othersfrom the natural gene. Combinations, as described, are also provided,e.g., combining the DCRS5 with the IL-12Rβ1, or their extracellularligand binding portions as ligand antagonists. Diagnostic utilities arealso clearly important, e.g., of polymorphic or other variants.

An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially pure, e.g., separated from othercomponents which naturally accompany a native sequence, such asribosomes, polymerases, and flanking genomic sequences from theoriginating species. The term embraces a nucleic acid sequence which hasbeen removed from its naturally occurring environment, and includesrecombinant or cloned DNA isolates, which are thereby distinguishablefrom naturally occurring compositions, and chemically synthesizedanalogs or analogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule,either completely or substantially pure.

An isolated nucleic acid will generally be a homogeneous composition ofmolecules, but will, in some embodiments, contain heterogeneity,preferably minor. This heterogeneity is typically found at the polymerends or portions not critical to a desired biological function oractivity.

A “recombinant” nucleic acid is typically defined either by its methodof production or its structure. In reference to its method ofproduction, e.g., a product made by a process, the process is use ofrecombinant nucleic acid techniques, e.g., involving human interventionin the nucleotide sequence. Typically this intervention involves invitro manipulation, although under certain circumstances it may involvemore classical animal breeding techniques. Alternatively, it can be anucleic acid made by generating a sequence comprising fusion of twofragments which are not naturally contiguous to each other, but is meantto exclude products of nature, e.g., naturally occurring mutants asfound in their natural state. Thus, e.g., products made by transformingcells with an unnaturally occurring vector is encompassed, as arenucleic acids comprising sequence derived using any syntheticoligonucleotide process. Such a process is often done to replace, e.g.,a codon with a redundant codon encoding the same or a conservative aminoacid, while typically introducing or removing a restriction enzymesequence recognition site, or for some structure-function analysis.Alternatively, the process is performed to join together nucleic acidsegments of desired functions to generate a single genetic entitycomprising a desired combination of functions not found in the commonlyavailable natural forms, e.g., encoding a fusion protein. Restrictionenzyme recognition sites are often the target of such artificialmanipulations, but other site specific targets, e.g., promoters, DNAreplication sites, regulation sequences, control sequences, or otheruseful features may be incorporated by design. A similar concept isintended for a recombinant, e.g., fusion, polypeptide. This will includea dimeric repeat or fusion of the DCRS5 with IL-12Rβ1 subunit.Specifically included are synthetic nucleic acids which, by genetic coderedundancy, encode equivalent polypeptides to fragments of DCRS5 andfusions of sequences from various different related molecules, e.g.,other cytokine family members.

A “fragment” in a nucleic acid context is a contiguous segment of atleast about 17 nucleotides, generally at least 21 nucleotides, moregenerally at least 25 nucleotides, ordinarily at least 30 nucleotides,more ordinarily at least 35 nucleotides, often at least 39 nucleotides,more often at least 45 nucleotides, typically at least 50 nucleotides,more typically at least 55 nucleotides, usually at least 60 nucleotides,more usually at least 66 nucleotides, preferably at least 72nucleotides, more preferably at least 79 nucleotides, and inparticularly preferred embodiments will be at least 85 or morenucleotides, including 90, 100, 120, 140, 160, 180, 200, etc. Typically,fragments of different genetic sequences can be compared to one anotherover appropriate length stretches, particularly defined segments such asthe domains described below.

A nucleic acid which codes for the DCRS5 will be particularly useful toidentify genes, mRNA, and cDNA species which code for itself or closelyrelated proteins, as well as DNAs which code for polymorphic, allelic,or other genetic variants, e.g., from different individuals or relatedspecies. Preferred probes for such screens are those regions of thereceptor which are conserved between different polymorphic variants orwhich contain nucleotides which lack specificity, and will preferably befull length or nearly so. In other situations, polymorphic variantspecific sequences will be more useful. Combinations of polymorphicvariants of DCRS5 with variants of IL-12Rβ1 may also be diagnosed.

This invention further covers recombinant nucleic acid molecules andfragments having a nucleic acid sequence identical to or highlyhomologous to the isolated DNA set forth herein. In particular, thesequences will often be operably linked to DNA segments which controltranscription, translation, and DNA replication. These additionalsegments typically assist in expression of the desired nucleic acidsegment.

Homologous, or highly identical, nucleic acid sequences, when comparedto one another, e.g., DCRS5 sequences, exhibit significant similarity.The standards for homology in nucleic acids are either measures forhomology generally used in the art by sequence comparison or based uponhybridization conditions. Comparative hybridization conditions aredescribed in greater detail below.

Substantial identity in the nucleic acid sequence comparison contextmeans either that the segments, or their complementary strands, whencompared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 60% of thenucleotides, generally at least 66%, ordinarily at least 71%, often atleast 76%, more often at least 80%, usually at least 84%, more usuallyat least 88%, typically at least 91%, more typically at least about 93%,preferably at least about 95%, more preferably at least about 96 to 98%or more, and in particular embodiments, as high at about 99% or more ofthe nucleotides, including, e.g., segments encoding structural domainsor other segments described. Alternatively, substantial identity willexist when the segments will hybridize under selective hybridizationconditions, to a strand or its complement, typically using a sequencederived from Table 1. Typically, selective hybridization will occur whenthere is at least about 55% homology over a stretch of at least about 14nucleotides, more typically at least about 65%, preferably at leastabout 75%, and more preferably at least about 90%. See, Kanehisa (1984)Nucl. Acids Res. 12:203-213, which is incorporated herein by reference.The length of homology comparison, as described, may be over longerstretches, and in certain embodiments will be over a stretch of at leastabout 17 nucleotides, generally at least about 20 nucleotides,ordinarily at least about 24 nucleotides, usually at least about 28nucleotides, typically at least about 32 nucleotides, more typically atleast about 40 nucleotides, preferably at least about 50 nucleotides,and more preferably at least about 75 to 100 or more nucleotides. Thisincludes, e.g., 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, etc.,and other lengths.

Stringent conditions, in referring to homology in the hybridizationcontext, will be stringent combined conditions of salt, temperature,organic solvents, and other parameters typically controlled inhybridization reactions. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C., more usually in excessof about 37° C., typically in excess of about 45° C., more typically inexcess of about 55° C., preferably in excess of about 65° C., and morepreferably in excess of about 70° C. Stringent salt conditions willordinarily be less than about 500 mM, usually less than about 400 mM,more usually less than about 300 mM, typically less than about 200 mM,preferably less than about 100 mM, and more preferably less than about80 mM, even down to less than about 50 or 20 mM. However, thecombination of parameters is much more important than the measure of anysingle parameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol.31:349-370, which is hereby incorporated herein by reference.

The isolated DNA can be readily modified by nucleotide substitutions,nucleotide deletions, nucleotide insertions, and inversions ofnucleotide stretches. These modifications result in novel DNA sequenceswhich encode this protein or its derivatives. These modified sequencescan be used to produce mutant proteins (muteins) or to enhance theexpression of variant species. Enhanced expression may involve geneamplification, increased transcription, increased translation, and othermechanisms. Such mutant DCRS5 inition of the DCRS5 as set forth above,but having an amino acid sequence which differs from that of othercytokine receptor-like proteins as found in nature, whether by way ofdeletion, substitution, or insertion. In particular, “site specificmutant DCRS5” encompasses a protein having substantial sequence identitywith a protein of Table 1, and typically shares most of the biologicalactivities or effects of the forms disclosed herein. Various naturalpolymorphic variant sequences will also be identified.

Although site specific mutation sites are predetermined, mutants neednot be site specific. Mammalian DCRS5 mutagenesis can be achieved bymaking amino acid insertions or deletions in the gene, coupled withexpression. Substitutions, deletions, insertions, or many combinationsmay be generated to arrive at a final construct. Insertions includeamino- or carboxy terminal fusions. Random mutagenesis can be conductedat a target codon and the expressed mammalian DCRS5 mutants can then bescreened for the desired activity, providing some aspect of astructure-activity relationship. Methods for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown in the art, e.g., by M13 primer mutagenesis. See also Sambrook, etal. (1989) and Ausubel, et al. (1987 and periodic Supplements).Particularly useful constructs will be extracellular portions of theDCRS5 associated with IL-12Rβ1 segments.

The mutations in the DNA normally should not place coding sequences outof reading frames and preferably will not create complementary regionsthat could hybridize to produce secondary mRNA structure such as loopsor hairpins.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence.

Polymerase chain reaction (PCR) techniques can often be applied inmutagenesis. Alternatively, mutagenesis primers are commonly usedmethods for generating defined mutations at predetermined sites. See,e.g., Innis, et al. (eds. 1990) PCR Protocols: A Guide to Methods andApplications Academic Press, San Diego, Calif.; and Dieffenbach andDveksler (1995; eds.) PCR Primer: A Laboratory Manual Cold Spring HarborPress, CSH, NY.

Certain embodiments of the invention are directed to combinationcompositions comprising the receptor sequences described. In otherembodiments, functional portions of the sequences may be joined toencode fusion proteins. In other forms, variants of the describedsequences may be substituted.

IV. Proteins, Peptides

As described above, the present invention encompasses primate DCRS5,e.g., whose sequences are disclosed in Table 1, and described above.Allelic and other variants are also contemplated, including, e.g.,fusion proteins combining portions of such sequences with others,including, e.g., IL-12Rβ1, epitope tags, and functional domains.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these primate or rodentproteins. A herterologous fusion protein is a fusion of proteins orsegments which are naturally not normally fused in the same manner.Thus, the fusion product of a DCRS5 with another cytokine receptor is acontinuous protein molecule having sequences fused in a typical peptidelinkage, typically made as a single translation product and exhibitingproperties, e.g., sequence or antigenicity, derived from each sourcepeptide. A similar concept applies to heterologous nucleic acidsequences. Combinations of various designated proteins into complexesare also provided.

In addition, new constructs may be made from combining similarfunctional or structural domains from other related proteins, e.g.,cytokine receptors or Toll-like receptors, including species variants.For example, ligand-binding or other segments may be “swapped” betweendifferent new fusion polypeptides or fragments. See, e.g., Cunningham,et al. (1989) Science 243:1330-1336; and O'Dowd, et al. (1988) J. Biol.Chem. 263:15985-15992, each of which is incorporated herein byreference. Thus, new chimeric polypeptides exhibiting new combinationsof specificities will result from the functional linkage ofreceptor-binding specificities. For example, the ligand binding domainsfrom other related receptor molecules may be added or substituted forother domains of this or related proteins. The resulting protein willoften have hybrid function and properties. For example, a fusion proteinmay include a targeting domain which may serve to provide sequesteringof the fusion protein to a particular subcellular organelle.

Candidate fusion partners and sequences can be selected from varioussequence data bases, e.g., GenBank, c/o IntelliGenetics, Mountain View,Calif.; and BCG, University of Wisconsin Biotechnology Computing Group,Madison, Wis., which are each incorporated herein by reference. Inparticular, combinations of polypeptide sequences provided in Tables 1and 3 are particularly preferred. Variant forms of the proteins may besubstituted in the described combinations.

The present invention particularly provides muteins which bindcytokine-like ligands, and/or which are affected in signal transduction.Structural alignment of human DCRS5 with other members of the cytokinereceptor family show conserved features/residues. See Table 3. Alignmentof the human DCRS5 sequence with other members of the cytokine receptorfamily indicates various structural and functionally shared features.See also, Bazan, et al. (1996) Nature 379:591; Lodi, et al. (1994)Science 263:1762-1766; Sayle and Milner-White (1995) TIBS 20:374-376;and Gronenberg, et al. (1991) Protein Engineering 4:263-269.

Substitutions with either mouse sequences or human sequences areparticularly preferred. Conversely, conservative substitutions away fromthe ligand binding interaction regions will probably preserve mostsignaling activities; and conservative substitutions away from theintracellular domains will probably preserve most ligand bindingproperties.

“Derivatives” of the primate DCRS5 include amino acid sequence mutants,glycosylation variants, metabolic derivatives and covalent oraggregative conjugates with other chemical moieties. Covalentderivatives can be prepared by linkage of functionalities to groupswhich are found in the DCRS5 amino acid side chains or at the N termini,e.g., by means which are well known in the art. These derivatives caninclude, without limitation, aliphatic esters or amides of the carboxylterminus, or of residues containing carboxyl side chains, O acylderivatives of hydroxyl group containing residues, and N acylderivatives of the amino terminal amino acid or amino group containingresidues, e.g., lysine or arginine. Acyl groups are selected from thegroup of alkyl moieties, including C3 to C18 normal alkyl, therebyforming alkanoyl aroyl species.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. Particularlypreferred means for accomplishing this are by exposing the polypeptideto glycosylating enzymes derived from cells which normally provide suchprocessing, e.g., mammalian glycosylation enzymes. Deglycosylationenzymes are also contemplated. Also embraced are versions of the sameprimary amino acid sequence which have other minor modifications,including phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine.

A major group of derivatives are covalent conjugates of the receptors orfragments thereof with other proteins of polypeptides. These derivativescan be synthesized in recombinant culture such as N terminal fusions orby the use of agents known in the art for their usefulness in crosslinking proteins through reactive side groups. Preferred derivatizationsites with cross linking agents are at free amino groups, carbohydratemoieties, and cysteine residues.

Fusion polypeptides between the receptors and other homologous orheterologous proteins are also provided. Homologous polypeptides may befusions between different receptors, resulting in, for instance, ahybrid protein exhibiting binding specificity for multiple differentcytokine ligands, or a receptor which may have broadened or weakenedspecificity of substrate effect. Likewise, heterologous fusions may beconstructed which would exhibit a combination of properties oractivities of the derivative proteins. Typical examples are fusions of areporter polypeptide, e.g., luciferase, with a segment or domain of areceptor, e.g., a ligand-binding segment, so that the presence orlocation of a desired ligand may be easily determined. See, e.g., Dull,et al., U.S. Pat. No. 4,859,609, which is hereby incorporated herein byreference. Other gene fusion partners include glutathione-S-transferase(GST), bacterial β-galactosidase, trpE, Protein A, β-lactamase, alphaamylase, alcohol dehydrogenase, and yeast alpha mating factor. See,e.g., Godowski, et al. (1988) Science 241:812-816. Labeled proteins willoften be substituted in the described combinations of proteins.Associations of the DCRS5 with the IL-12Rβ1 are particularlysignificant, as described.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra, Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence.

Such polypeptides may also have amino acid residues which have beenchemically modified by phosphorylation, sulfonation, biotinylation, orthe addition or removal of other moieties, particularly those which havemolecular shapes similar to phosphate groups. In some embodiments, themodifications will be useful labeling reagents, or serve as purificationtargets, e.g., affinity ligands.

Fusion proteins will typically be made by either recombinant nucleicacid methods or by synthetic polypeptide methods. Techniques for nucleicacid manipulation and expression are described generally, for example,in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2ded.), Vols. 1 3, Cold Spring Harbor Laboratory, and Ausubel, et al.(eds. 1987 and periodic supplements) Current Protocols in MolecularBiology, Greene/Wiley, New York, which are each incorporated herein byreference. Techniques for synthesis of polypeptides are described, forexample, in Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156;Merrifield (1986) Science 232: 341-347; and Atherton, et al. (1989)Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford;each of which is incorporated herein by reference. See also Dawson, etal. (1994) Science 266:776-779 for methods to make larger polypeptides.

This invention also contemplates the use of derivatives of a DCRS5 otherthan variations in amino acid sequence or glycosylation. Suchderivatives may involve covalent or aggregative association withchemical moieties. These derivatives generally fall into three classes:(1) salts, (2) side chain and terminal residue covalent modifications,and (3) adsorption complexes, e.g., with cell membranes. Such covalentor aggregative derivatives are useful as immunogens, as reagents inimmunoassays, or in purification methods such as for affinitypurification of a receptor or other binding molecule, e.g., an antibody.For example, a cytokine ligand can be immobilized by covalent bonding toa solid support such as cyanogen bromide activated Sepharose, by methodswhich are well known in the art, or adsorbed onto polyolefin surfaces,with or without glutaraldehyde cross linking, for use in the assay orpurification of a cytokine receptor, antibodies, or other similarmolecules. The ligand can also be labeled with a detectable group, e.g.,radioiodinated by the chloramine T procedure, covalently bound to rareearth chelates, or conjugated to another fluorescent moiety for use indiagnostic assays.

A combination, e.g., including a DCRS5, of this invention can be used asan immunogen for the production of antisera or antibodies specific,e.g., capable of distinguishing between other cytokine receptor familymembers, for the combinations described. The complexes can be used toscreen monoclonal antibodies or antigen-binding fragments prepared byimmunization with various forms of impure preparations containing theprotein. In particular, the term “antibodies” also encompasses antigenbinding fragments of natural antibodies, e.g., Fab, Fab2, Fv, etc. Thepurified DCRS5 can also be used as a reagent to detect antibodiesgenerated in response to the presence of elevated levels of expression,or immunological disorders which lead to antibody production to theendogenous receptor. Additionally, DCRS5 fragments may also serve asimmunogens to produce the antibodies of the present invention, asdescribed immediately below. For example, this invention contemplatesantibodies having binding affinity to or being raised against the aminoacid sequences shown in Table 1, fragments thereof, or varioushomologous peptides. In particular, this invention contemplatesantibodies having binding affinity to, or having been raised against,specific fragments which are predicted to be, or actually are, exposedat the exterior protein surface of the native DCRS5. Complexes ofcombinations of proteins will also be useful, and antibody preparationsthereto can be made.

In certain other embodiments, soluble constructs, e.g., of theextracellular ligand binding segments of the DCRS5 with the IL-12Rβ1 maybe binding compositions for the ligand and may be useful as eitherligand antagonists, or as antigens to block ligand mediated signaling.Such may be useful either diagnostically, e.g., for histology labelingfor ligand, or therapeutically, e.g., as ligand antagonists.

The blocking of physiological response to the receptor ligands mayresult from the inhibition of binding of the ligand to the receptor,likely through competitive inhibition. Thus, in vitro assays of thepresent invention will often use antibodies or antigen binding segmentsof these antibodies, soluble receptor constructs, or fragments attachedto solid phase substrates. These assays will also allow for thediagnostic determination of the effects of either ligand binding regionmutations and modifications, or other mutations and modifications, e.g.,which affect signaling or enzymatic function.

This invention also contemplates the use of competitive drug screeningassays, e.g., where neutralizing antibodies to the receptor complexes orfragments compete with a test compound for binding to a ligand or otherantibody. In this manner, the neutralizing antibodies or fragments canbe used to detect the presence of a polypeptide which shares one or morebinding sites to a receptor and can also be used to occupy binding siteson a receptor that might otherwise bind a ligand. Soluble receptorconstructs combining the extracellular, or ligand binding, domains ofthe DCRS5 with the IL-12Rβ1, may be useful antagonists for competitivebinding of p40/IL-B30 ligand.

V. Making Nucleic Acids and Protein

DNA which encodes the protein or fragments thereof can be obtained bychemical synthesis, screening cDNA libraries, or by screening genomiclibraries prepared from a wide variety of cell lines or tissue samples.Natural sequences can be isolated using standard methods and thesequences provided herein, e.g., in Table 1. Other species counterpartscan be identified by hybridization techniques, or by various PCRtechniques, combined with or by searching in sequence databases, e.g.,GenBank.

This DNA can be expressed in a wide variety of host cells for thesynthesis of a full length receptor or fragments which can in turn, forexample, be used to generate polyclonal or monoclonal antibodies; forbinding studies; for construction and expression of modified ligandbinding or kinase/phosphatase domains; and for structure/functionstudies. Variants or fragments can be expressed in host cells that aretransformed or transfected with appropriate expression vectors. Thesemolecules can be substantially free of protein or cellular contaminants,other than those derived from the recombinant host, and therefore areparticularly useful in pharmaceutical compositions when combined with apharmaceutically acceptable carrier and/or diluent. The protein, orportions thereof, may be expressed as fusions with other proteins.Combinations of the described proteins, or nucleic acids encoding them,are particularly interesting.

Expression vectors are typically self replicating DNA or RNA constructscontaining the desired receptor gene, its fragments, or combinationgenes, usually operably linked to suitable genetic control elements thatare recognized in a suitable host cell. These control elements arecapable of effecting expression within a suitable host. Multiple genesmay be coordinately expressed, and may be on a polycistronic message.The specific type of control elements necessary to effect expressionwill depend upon the eventual host cell used. Generally, the geneticcontrol elements can include a prokaryotic promoter system or aeukaryotic promoter expression control system, and typically include atranscriptional promoter, an optional operator to control the onset oftranscription, transcription enhancers to elevate the level of mRNAexpression, a sequence that encodes a suitable ribosome binding site,and sequences that terminate transcription and translation. Expressionvectors also usually contain an origin of replication that allows thevector to replicate independently of the host cell.

The vectors of this invention include those which contain DNA whichencodes a combination of proteins, as described, or a biologicallyactive equivalent polypeptide. The DNA can be under the control of aviral promoter and can encode a selection marker. This invention furthercontemplates use of such expression vectors which are capable ofexpressing eukaryotic cDNAs coding for such proteins in a prokaryotic oreukaryotic host, where the vector is compatible with the host and wherethe eukaryotic cDNAs are inserted into the vector such that growth ofthe host containing the vector expresses the cDNAs in question. Usually,expression vectors are designed for stable replication in their hostcells or for amplification to greatly increase the total number ofcopies of the desirable gene(s) per cell. It is not always necessary torequire that an expression vector replicate in a host cell, e.g., it ispossible to effect transient expression of the protein or its fragmentsin various hosts using vectors that do not contain a replication originthat is recognized by the host cell. It is also possible to use vectorsthat cause integration of the protein encoding portions into the hostDNA by recombination.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles which enable theintegration of DNA fragments into the genome of the host. Expressionvectors are specialized vectors which contain genetic control elementsthat effect expression of operably linked genes. Plasmids are the mostcommonly used form of vector but all other forms of vectors which servean equivalent function and which are, or become, known in the art aresuitable for use herein. See, e.g., Pouwels, et al. (1985 andSupplements) Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., andRodriguez, et al. (eds. 1988) Vectors: A Survey of Molecular CloningVectors and Their Uses, Buttersworth, Boston, which are incorporatedherein by reference.

Transformed cells are cells, preferably mammalian, that have beentransformed or transfected with vectors constructed using recombinantDNA techniques. Transformed host cells usually express the desiredproteins, but for purposes of cloning, amplifying, and manipulating itsDNA, do not need to express the subject proteins. This invention furthercontemplates culturing transformed cells in a nutrient medium, thuspermitting the proteins to accumulate. The proteins can be recovered,either from the culture or, in certain instances, from the culturemedium.

For purposes of this invention, nucleic sequences are operably linkedwhen they are functionally related to each other. For example, DNA for apresequence or secretory leader is operably linked to a polypeptide ifit is expressed as a preprotein or participates in directing thepolypeptide to the cell membrane or in secretion of the polypeptide. Apromoter is operably linked to a coding sequence if it controls thetranscription of the polypeptide; a ribosome binding site is operablylinked to a coding sequence if it is positioned to permit translation.Usually, operably linked means contiguous and in reading frame, however,certain genetic elements such as repressor genes are not contiguouslylinked but still bind to operator sequences that in turn controlexpression.

Suitable host cells include prokaryotes, lower eukaryotes, and highereukaryotes. Prokaryotes include both gram negative and gram positiveorganisms, e.g., E. coli and B. subtilis. Lower eukaryotes includeyeasts, e.g., S. cerevisiae and Pichia, and species of the genusDictyostelium. Higher eukaryotes include established tissue culture celllines from animal cells, both of non mammalian origin, e.g., insectcells, and birds, and of mammalian origin, e.g., human, primates, androdents.

Prokaryotic host vector systems include a wide variety of vectors formany different species. As used herein, E. coli and its vectors will beused generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 ormany of its derivatives. Vectors that can be used to express thereceptor or its fragments include, but are not limited to, such vectorsas those containing the lac promoter (pUC series); trp promoter (pBR322trp); Ipp promoter (the pIN series); lambda pP or pR promoters (pOTS);or hybrid promoters such as ptac (pDR540). See Brosius, et al. (1988)“Expression Vectors Employing Lambda, and Ipp derived Promoters”, inVectors: A Survey of Molecular Cloning Vectors and Their Uses, (eds.Rodriguez and Denhardt), Buttersworth, Boston, Chapter 10, pp. 205 236,which is incorporated herein by reference.

Lower eukaryotes, e.g., yeasts and Dictyostelium, may be transformedwith DCRS5 sequence containing vectors. For purposes of this invention,the most common lower eukaryotic host is the baker's yeast,Saccharomyces cerevisiae. It will be used to generically represent lowereukaryotes although a number of other strains and species are alsoavailable. Yeast vectors typically consist of a replication origin(unless of the integrating type), a selection gene, a promoter, DNAencoding the receptor or its fragments, and sequences for translationtermination, polyadenylation, and transcription termination. Suitableexpression vectors for yeast include such constitutive promoters as 3phosphoglycerate kinase and various other glycolytic enzyme genepromoters or such inducible promoters as the alcohol dehydrogenase 2promoter or metallothionine promoter. Suitable vectors includederivatives of the following types: self replicating low copy number(such as the YRp series), self replicating high copy number (such as theYEp series); integrating types (such as the YIp series), or minichromosomes (such as the YCp series).

Higher eukaryotic tissue culture cells are normally the preferred hostcells for expression of the functionally active interleukin or receptorproteins. In principle, many higher eukaryotic tissue culture cell linesare workable, e.g., insect baculovirus expression systems, whether froman invertebrate or vertebrate source. However, mammalian cells arepreferred. Transformation or transfection and propagation of such cellshas become a routine procedure. Examples of useful cell lines includeHeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney(BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS)cell lines. Expression vectors for such cell lines usually include anorigin of replication, a promoter, a translation initiation site, RNAsplice sites (if genomic DNA is used), a polyadenylation site, and atranscription termination site. These vectors also usually contain aselection gene or amplification gene. Suitable expression vectors may beplasmids, viruses, or retroviruses carrying promoters derived, e.g.,from such sources as from adenovirus, SV40, parvoviruses, vacciniavirus, or cytomegalovirus. Representative examples of suitableexpression vectors include pCDNA1; pCD, see Okayama, et al. (1985) Mol.Cell. Biol. 5:1136 1142; pMC1 neo PolyA, see Thomas, et al. (1987) Cell51:503 512; and a baculovirus vector such as pAC 373 or pAC 610.

For secreted proteins and some membrane proteins, an open reading frameusually encodes a polypeptide that consists of a mature or secretedproduct covalently linked at its N-terminus to a signal peptide. Thesignal peptide is cleaved prior to secretion of the mature, or active,polypeptide. The cleavage site can be predicted with a high degree ofaccuracy from empirical rules, e.g., von-Heijne (1986) Nucleic AcidsResearch 14:4683-4690 and Nielsen, et al. (1997) Protein Eng. 10:1-12,and the precise amino acid composition of the signal peptide often doesnot appear to be critical to its function, e.g., Randall, et al. (1989)Science 243:1156-1159; Kaiser et al. (1987) Science 235:312-317. Themature proteins of the invention can be readily determined usingstandard methods.

It will often be desired to express these polypeptides in a system whichprovides a specific or defined glycosylation pattern. In this case, theusual pattern will be that provided naturally by the expression system.However, the pattern will be modifiable by exposing the polypeptide,e.g., an unglycosylated form, to appropriate glycosylating proteinsintroduced into a heterologous expression system. For example, thereceptor gene may be co-transformed with one or more genes encodingmammalian or other glycosylating enzymes. Using this approach, certainmammalian glycosylation patterns will be achievable in prokaryote orother cells. Expression in prokaryote cells will typically lead tounglycosylated forms of protein.

The source of DCRS5 can be a eukaryotic or prokaryotic host expressingrecombinant DCRS5, such as is described above. The source can also be acell line, but other mammalian cell lines are also contemplated by thisinvention, with the preferred cell line being from the human species.

Now that the sequences are known, the primate DCRS5, fragments, orderivatives thereof can be prepared by conventional processes forsynthesizing peptides. These include processes such as are described inStewart and Young (1984) Solid Phase Peptide Synthesis, Pierce ChemicalCo., Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice ofPeptide Synthesis, Springer Verlag, New York; and Bodanszky (1984) ThePrinciples of Peptide Synthesis, Springer Verlag, New York; all of eachwhich are incorporated herein by reference. For example, an azideprocess, an acid chloride process, an acid anhydride process, a mixedanhydride process, an active ester process (for example, p nitrophenylester, N hydroxysuccinimide ester, or cyanomethyl ester), acarbodiimidazole process, an oxidative reductive process, or adicyclohexylcarbodiimide (DCCD) additive process can be used. Solidphase and solution phase syntheses are both applicable to the foregoingprocesses. Similar techniques can be used with partial DCRS5 sequences.

The DCRS5 proteins, fragments, or derivatives are suitably prepared inaccordance with the above processes as typically employed in peptidesynthesis, generally either by a so called stepwise process whichcomprises condensing an amino acid to the terminal amino acid, one byone in sequence, or by coupling peptide fragments to the terminal aminoacid. Amino groups that are not being used in the coupling reactiontypically must be protected to prevent coupling at an incorrectlocation.

If a solid phase synthesis is adopted, the C terminal amino acid isbound to an insoluble carrier or support through its carboxyl group. Theinsoluble carrier is not particularly limited as long as it has abinding capability to a reactive carboxyl group. Examples of suchinsoluble carriers include halomethyl resins, such as chloromethyl resinor bromomethyl resin, hydroxymethyl resins, phenol resins, tertalkyloxycarbonylhydrazidated resins, and the like.

An amino group protected amino acid is bound in sequence throughcondensation of its activated carboxyl group and the reactive aminogroup of the previously formed peptide or chain, to synthesize thepeptide step by step. After synthesizing the complete sequence, thepeptide is split off from the insoluble carrier to produce the peptide.This solid phase approach is generally described by Merrifield, et al.(1963) in J. Am. Chem. Soc. 85:2149 2156, which is incorporated hereinby reference.

The prepared protein and fragments thereof can be isolated and purifiedfrom the reaction mixture by means of peptide separation, e.g., byextraction, precipitation, electrophoresis, various forms ofchromatography, immunoaffinity, and the like. The receptors of thisinvention can be obtained in varying degrees of purity depending upondesired uses. Purification can be accomplished by use of the proteinpurification techniques disclosed herein, see below, or by the use ofthe antibodies herein described in methods of immunoabsorbant affinitychromatography. This immunoabsorbant affinity chromatography is carriedout by first linking the antibodies to a solid support and thencontacting the linked antibodies with solubilized lysates of appropriatecells, lysates of other cells expressing the receptor, or lysates orsupernatants of cells producing the protein as a result of DNAtechniques, see below.

Generally, the purified protein will be at least about 40% pure,ordinarily at least about 50% pure, usually at least about 60% pure,typically at least about 70% pure, more typically at least about 80%pure, preferable at least about 90% pure and more preferably at leastabout 95% pure, and in particular embodiments, 97%-99% or more. Puritywill usually be on a weight basis, but can also be on a molar basis.Different assays will be applied as appropriate. Individual proteins maybe purified and thereafter combined.

VI. Antibodies

Antibodies can be raised to the various mammalian, e.g., primate DCRS5proteins and fragments thereof, both in naturally occurring native formsand in their recombinant forms, the difference being that antibodies tothe active receptor are more likely to recognize epitopes which are onlypresent in the native conformations. Antibodies recognizing epitopespresented by the combination, e.g., functionally, of the DCRS5 with theIL-12Rβ1 are also contemplated. Denatured antigen detection can also beuseful in, e.g., Western analysis. Anti-idiotypic antibodies are alsocontemplated, which would be useful as agonists or antagonists of anatural receptor or an antibody.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of the protein can be raised byimmunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective protein, or screened for agonistic orantagonistic activity. These monoclonal antibodies will usually bindwith at least a KD of about 1 mM, more usually at least about 300 μM,typically at least about 100 μM, more typically at least about 30 μM,preferably at least about 10 μM, and more preferably at least about 3 μMor better.

The antibodies, including antigen binding fragments, of this inventioncan have significant diagnostic or therapeutic value. They can be potentantagonists that bind to the receptor and inhibit binding to ligand orinhibit the ability of the receptor to elicit a biological response,e.g., act on its substrate. They also can be useful as non neutralizingantibodies and can be coupled to toxins or radionuclides to bindproducing cells, or cells localized to the source of the interleukin.Further, these antibodies can be conjugated to drugs or othertherapeutic agents, either directly or indirectly by means of a linker.

The antibodies of this invention can also be useful in diagnosticapplications. As capture or non neutralizing antibodies, they might bindto the receptor without inhibiting ligand or substrate binding. Asneutralizing antibodies, they can be useful in competitive bindingassays. They will also be useful in detecting or quantifying ligand.They may be used as reagents for Western blot analysis, or forimmunoprecipitation or immunopurification of the respective protein.Likewise, nucleic acids and proteins may be immobilized to solidsubstrates for affinity purification or detection methods. Thesubstrates may be, e.g., solid resin beads or sheets of plastic.

Protein fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. Mammalian cytokine receptors and fragments may be fused orcovalently linked to a variety of immunogens, such as keyhole limpethemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbiology,Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962)Specificity of Serological Reactions, Dover Publications, New York; andWilliams, et al. (1967) Methods in Immunology and Immunochemistry, Vol.1, Academic Press, New York; each of which are incorporated herein byreference, for descriptions of methods of preparing polyclonal antisera.A typical method involves hyperimmunization of an animal with anantigen. The blood of the animal is then collected shortly after therepeated immunizations and the gamma globulin is isolated.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.), Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual,CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.) Academic Press, New York; and particularly in Kohler andMilstein (1975) in Nature 256: 495 497, which discusses one method ofgenerating monoclonal antibodies. Each of these references isincorporated herein by reference. Summarized briefly, this methodinvolves injecting an animal with an immunogen. The animal is thensacrificed and cells taken from its spleen, which are then fused withmyeloma cells. The result is a hybrid cell or “hybridoma” that iscapable of reproducing in vitro. The population of hybridomas is thenscreened to isolate individual clones, each of which secrete a singleantibody species to the immunogen. In this manner, the individualantibody species obtained are the products of immortalized and clonedsingle B cells from the immune animal generated in response to aspecific site recognized on the immunogenic substance.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse, et al. (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275-1281; and Ward, et al.(1989) Nature 341:544-546, each of which is hereby incorporated hereinby reference. The polypeptides and antibodies of the present inventionmay be used with or without modification, including chimeric orhumanized antibodies. Frequently, the polypeptides and antibodies willbe labeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmoieties, chemiluminescent moieties, magnetic particles, and the like.Patents, teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant or chimeric immunoglobulins may beproduced, see Cabilly, U.S. Pat. No. 4,816,567; or made in transgenicmice, see Mendez, et al. (1997) Nature Genetics 15:146-156. Thesereferences are incorporated herein by reference.

The antibodies of this invention can also be used for affinitychromatography in isolating the DCRS5 proteins or peptides. Columns canbe prepared where the antibodies are linked to a solid support, e.g.,particles, such as agarose, Sephadex, or the like, where a cell lysatemay be passed through the column, the column washed, followed byincreasing concentrations of a mild denaturant, whereby the purifiedprotein will be released. Alternatively, the protein may be used topurify antibody. Appropriate cross absorptions or depletions may beapplied.

The antibodies may also be used to screen expression libraries forparticular expression products. Usually the antibodies used in such aprocedure will be labeled with a moiety allowing easy detection ofpresence of antigen by antibody binding.

Antibodies raised against a cytokine receptor will also be used to raiseanti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of theprotein or cells which express the protein. They also will be useful asagonists or antagonists of the ligand, which may be competitive receptorinhibitors or substitutes for naturally occurring ligands. Certainantibodies to receptor subunits or combinations may serve as activatingantibodies, which may effect signaling thereby serving, e.g., as ligandagonists.

A cytokine receptor protein that specifically binds to or that isspecifically immunoreactive with an antibody generated against a definedimmunogen, such as an immunogen consisting of the amino acid sequence ofSEQ ID NO: 2, is typically determined in an immunoassay. The immunoassaytypically uses a polyclonal antiserum which was raised, e.g., to aprotein of SEQ ID NO: 2. This antiserum is selected to have lowcrossreactivity against other cytokine receptor family members, e.g.,IL-12Rβ2 receptor subunit or IL-6 receptor subunit gp 130, preferablyfrom the same species, and any such crossreactivity is removed byimmunoabsorption prior to use in the immunoassay.

In order to produce antisera for use in an immunoassay, the protein,e.g., of SEQ ID NO: 2, is isolated as described herein. For example,recombinant protein may be produced in a mammalian cell line. Anappropriate host, e.g., an inbred strain of mice such as Balb/c, isimmunized with the selected protein, typically using a standardadjuvant, such as Freund's adjuvant, and a standard mouse immunizationprotocol (see Harlow and Lane, supra). Alternatively, a syntheticpeptide derived from the sequences disclosed herein and conjugated to acarrier protein can be used an immunogen. Polyclonal sera are collectedand titered against the immunogen protein in an immunoassay, e.g., asolid phase immunoassay with the immunogen immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁴ or greater are selectedand tested for their cross reactivity against other cytokine receptorfamily members, e.g., gp130 or IL-12Rβ1 using a competitive bindingimmunoassay such as the one described in Harlow and Lane, supra, atpages 570-573. Preferably at least two cytokine receptor family membersare used in this determination. These cytokine receptor family memberscan be produced as recombinant proteins and isolated using standardmolecular biology and protein chemistry techniques as described herein.

Immunoassays in the competitive binding format can be used for thecrossreactivity determinations. For example, the protein of SEQ ID NO: 2can be immobilized to a solid support. Proteins added to the assaycompete with the binding of the antisera to the immobilized antigen. Theability of the above proteins to compete with the binding of theantisera to the immobilized protein is compared to the proteins, e.g.,of gp130 or IL-12Rβ2. The percent crossreactivity for the above proteinsis calculated, using standard calculations. Those antisera with lessthan 10% crossreactivity with each of the proteins listed above areselected and pooled. The cross-reacting antibodies are then removed fromthe pooled antisera by immunoabsorption with the above-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein tothe immunogen protein (e.g., the DCRS5 like protein of SEQ ID NO: 2). Inorder to make this comparison, the two proteins are each assayed at awide range of concentrations and the amount of each protein required toinhibit 50% of the binding of the antisera to the immobilized protein isdetermined. If the amount of the second protein required is less thantwice the amount of the protein of the selected protein or proteins thatis required, then the second protein is said to specifically bind to anantibody generated to the immunogen.

It is understood that these cytokine receptor proteins are members of afamily of homologous proteins that comprise many identified genes. For aparticular gene product, such as the DCRS5, the term refers not only tothe amino acid sequences disclosed herein, but also to other proteinsthat are allelic, non-allelic, or species variants. It is alsounderstood that the terms include nonnatural mutations introduced bydeliberate mutation using conventional recombinant technology such assingle site mutation, or by excising short sections of DNA encoding therespective proteins, or by substituting new amino acids, or adding newamino acids. Such minor alterations typically will substantiallymaintain the immunoidentity of the original molecule and/or itsbiological activity. Thus, these alterations include proteins that arespecifically immunoreactive with a designated naturally occurring DCRS5protein. The biological properties of the altered proteins can bedetermined by expressing the protein in an appropriate cell line andmeasuring the appropriate effect, e.g., upon transfected lymphocytes.Particular protein modifications considered minor would includeconservative substitution of amino acids with similar chemicalproperties, as described above for the cytokine receptor family as awhole. By aligning a protein optimally with the protein of the cytokinereceptors and by using the conventional immunoassays described herein todetermine immunoidentity, one can determine the protein compositions ofthe invention.

Moreover, antibodies against the receptor subunits may serve tosterically block ligand binding to the functional receptor. Suchantibodies may be raised to either subunit alone, or to the combinationof DCRS5 with IL-12Rβ1. Antibody antagonists would result.

VII. Kits, Diagnosis, and Quantitation

Both naturally occurring and recombinant forms of the cytokine receptorlike molecules of this invention are particularly useful in kits andassay methods. For example, these methods would also be applied toscreening for binding activity, e.g., ligands for these proteins.Several methods of automating assays have been developed in recent yearsso as to permit screening of tens of thousands of compounds per year.See, e.g., a BIOMEK automated workstation, Beckman Instruments, PaloAlto, Calif., and Fodor, et al. (1991) Science 251:767-773, which isincorporated herein by reference. The latter describes means for testingbinding by a plurality of defined polymers synthesized on a solidsubstrate. The development of suitable assays to screen for a ligand oragonist/antagonist homologous proteins can be greatly facilitated by theavailability of large amounts of purified, soluble cytokine receptors inan active state such as is provided by this invention.

Purified DCRS5 can be coated directly onto plates for use in theaforementioned ligand screening techniques. However, non neutralizingantibodies to these proteins can be used as capture antibodies toimmobilize the respective receptor on the solid phase, useful, e.g., indiagnostic uses.

This invention also contemplates use of DCRS5, fragments thereof,peptides, and their fusion products in a variety of diagnostic kits andmethods for detecting the presence of the protein or its ligand.Alternatively, or additionally, antibodies against the molecules may beincorporated into the kits and methods. Typically the kit will have acompartment containing either a DCRS5 peptide or gene segment or areagent which recognizes one or the other. Typically, recognitionreagents, in the case of peptide, would be a receptor or antibody, or inthe case of a gene segment, would usually be a hybridization probe.Other kit components may include other proteins or reagents related tothe p40, IL-B30, or IL-12Rβ1 polypeptides of the ligand/receptorpairing.

A preferred kit for determining the concentration of DCRS5 in a samplewould typically comprise a labeled compound, e.g., ligand or antibody,having known binding affinity for DCRS5, a source of DCRS5 (naturallyoccurring or recombinant) as a positive control, and a means forseparating the bound from free labeled compound, for example a solidphase for immobilizing the DCRS5 in the test sample. Compartmentscontaining reagents, and instructions, will normally be provided.Appropriate nucleic acid or protein containing kits are also provided.

Antibodies, including antigen binding fragments, specific for mammalianDCRS5 or a peptide fragment, or receptor fragments are useful indiagnostic applications to detect the presence of elevated levels ofligand and/or its fragments. Diagnostic assays may be homogeneous(without a separation step between free reagent and antibody-antigencomplex) or heterogeneous (with a separation step). Various commercialassays exist, such as radioimmunoassay (RIA), enzyme linkedimmunosorbent assay (ELISA), enzyme immunoassay (EIA), enzyme multipliedimmunoassay technique (EMIT), substrate labeled fluorescent immunoassay(SLFIA) and the like. For example, unlabeled antibodies can be employedby using a second antibody which is labeled and which recognizes theantibody to a cytokine receptor or to a particular fragment thereof.These assays have also been extensively discussed in the literature.See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH.,and Coligan (ed. 1991 and periodic supplements) Current Protocols InImmunology Greene/Wiley, New York.

Anti-idiotypic antibodies may have similar use to serve as agonists orantagonists of cytokine receptors. These should be useful as therapeuticreagents under appropriate circumstances.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody, or labeled ligand is provided.This is usually in conjunction with other additives, such as buffers,stabilizers, materials necessary for signal production such assubstrates for enzymes, and the like. Preferably, the kit will alsocontain instructions for proper use and disposal of the contents afteruse. Typically the kit has compartments for each useful reagent, andwill contain instructions for proper use and disposal of reagents.Desirably, the reagents are provided as a dry lyophilized powder, wherethe reagents may be reconstituted in an aqueous medium havingappropriate concentrations for performing the assay.

The aforementioned constituents of the diagnostic assays may be usedwithout modification or may be modified in a variety of ways. Forexample, labeling may be achieved by covalently or non covalentlyjoining a moiety which directly or indirectly provides a detectablesignal. In many of these assays, a test compound, cytokine receptor, orantibodies thereto can be labeled either directly or indirectly.Possibilities for direct labeling include label groups: radiolabels suchas ¹²⁵I, enzymes (U.S. Pat. No. 3,645,090) such as peroxidase andalkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475)capable of monitoring the change in fluorescence intensity, wavelengthshift, or fluorescence polarization. Both of the patents areincorporated herein by reference. Possibilities for indirect labelinginclude biotinylation of one constituent followed by binding to avidincoupled to one of the above label groups.

There are also numerous methods of separating the bound from the freeligand, or alternatively the bound from the free test compound. Thecytokine receptor can be immobilized on various matrixes followed bywashing. Suitable matrices include plastic such as an ELISA plate,filters, and beads. Methods of immobilizing the receptor to a matrixinclude, without limitation, direct adhesion to plastic, use of acapture antibody, chemical coupling, and biotin avidin. The last step inthis approach involves the precipitation of antibody/antigen complex byany of several methods including those utilizing, e.g., an organicsolvent such as polyethylene glycol or a salt such as ammonium sulfate.Other suitable separation techniques include, without limitation, thefluorescein antibody magnetizable particle method described in Rattle,et al. (1984) Clin. Chem. 30(9):1457 1461, and the double antibodymagnetic particle separation as described in U.S. Pat. No. 4,659,678,each of which is incorporated herein by reference.

The methods for linking protein or fragments to various labels have beenextensively reported in the literature and do not require detaileddiscussion here. Many of the techniques involve the use of activatedcarboxyl groups either through the use of carbodiimide or active estersto form peptide bonds, the formation of thioethers by reaction of amercapto group with an activated halogen such as chloroacetyl, or anactivated olefin such as maleimide, for linkage, or the like. Fusionproteins will also find use in these applications.

Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence ofan cytokine receptor. These sequences can be used as probes fordetecting levels of the respective cytokine receptor in patientssuspected of having an immunological disorder. The preparation of bothRNA and DNA nucleotide sequences, the labeling of the sequences, and thepreferred size of the sequences has received ample description anddiscussion in the literature. Normally an oligonucleotide probe shouldhave at least about 14 nucleotides, usually at least about 18nucleotides, and the polynucleotide probes may be up to severalkilobases. Various labels may be employed, most commonly radionuclides,particularly ³²P. However, other techniques may also be employed, suchas using biotin modified nucleotides for introduction into apolynucleotide. The biotin then serves as the site for binding to avidinor antibodies, which may be labeled with a wide variety of labels, suchas radionuclides, fluorescers, enzymes, or the like. Alternatively,antibodies may be employed which can recognize specific duplexes,including DNA duplexes, RNA duplexes, DNA RNA hybrid duplexes, or DNAprotein duplexes. The antibodies in turn may be labeled and the assaycarried out where the duplex is bound to a surface, so that upon theformation of duplex on the surface, the presence of antibody bound tothe duplex can be detected. The use of probes to the novel anti senseRNA may be carried out in conventional techniques such as nucleic acidhybridization, plus and minus screening, recombinational probing, hybridreleased translation (HRT), and hybrid arrested translation (HART). Thisalso includes amplification techniques such as polymerase chain reaction(PCR).

Diagnostic kits which also test for the qualitative or quantitativepresence of other markers are also contemplated. Diagnosis or prognosismay depend on the combination of multiple indications used as markers.Thus, kits may test for combinations of markers. See, e.g., Viallet, etal. (1989) Progress in Growth Factor Res. 1:89-97. Detection ofpolymorphic variations, which may reflect functional receptor signalingdifferences, may be useful in determining therapeutic strategy.Variations which reflect greater or lesser response to ligand may allowsubsetting of responsive/non-responsive patient pools.

VIII. Therapeutic Utility

This invention provides reagents with significant therapeutic value.See, e.g., Levitzki (1996) Curr. Opin. Cell Biol. 8:239-244. Thecytokine receptors (naturally occurring or recombinant), fragmentsthereof, mutein receptors, and antibodies, along with compoundsidentified as having binding affinity to the receptors or antibodies,should be useful in the treatment of conditions exhibiting abnormalexpression of the receptors or their ligands. Such abnormality willtypically be manifested by immunological disorders. See WO 01/18051,which is incorporated herein by reference. Additionally, this inventionshould provide therapeutic value in various diseases or disordersassociated with abnormal expression or abnormal triggering of responseto the ligand. For example, the p40/IL B30 ligand has been suggested tobe involved in development of cell mediated immunity, e.g., anti-tumoractivity, mounting of humoral and cellular immunity, and antiviraleffects. In particular, the ligand appears to activate NK and T cells.Therapy may be combined with IL-18, IL-12, TNF, IFNγ, radiation/chemotherapy, adjuvants, or antitumor, antiviral, or antifungal compounds.

Conversely, antagonists, which may be combined with antagonists of TNF,IFNγ, IL-18, or IL-12, or with IL-10 or steroids, may be indicated inchronic Th1 mediated diseases, autoimmunity, or transplant and/orrejection situations, multiple sclerosis, psoriasis, chronicinflammatory conditions, rheumatoid arthritis, osteoarthritis, orinflammatory bowel diseases. Antagonists may take the form of antibodiesagainst the receptor subunits, soluble receptor constructs, or antisensenucleic acids to one or more of the receptor subunits. The matching ofthe p40/IL-B30 ligand with receptor subunits DCRS5 and IL-12Rβ1 providesinsight into indications for use of the agonists and antagonists.

Therapeutically, based on the p40/IL-B30 activities described,antagonists of the cytokine may be effected, e.g., by soluble DCRS5,with or without soluble IL-12Rβ1, or antibodies to either receptorsubunit. Antagonists my be useful as inhibitors of undesirable immune orinflammatory responses, to target memory T cells, or in combination withIL-12/IL-12R antagonists, or other anti-inflammatories orimmunosuppressants. Clinical indications may be chronic inflammation ortransplant situations. Various polymorphisms may enhance or decreasereceptor function, and if dominant, might be useful as therapeutics.Identification of such variants may allow subsetting of responsive ornonresponsive patient pools. The reagents may be useful as detecting orlabeling reagents or ablative reagents for memory T cells and/or NKcells.

Gene therapy may render desired cell populations response to p40/IL-B30ligand, e.g., as adjuvants for tumor immunotherapy, to facilitateactivation of tumor infiltrating lymphocytes, T cells, or NK cells.Antisense strategies may be applied, e.g., to prevent receptorresponsiveness.

Various abnormal conditions are known in cell types shown to produceboth IL-12 p40 and/or IL-B30 mRNA by Northern blot analysis. See Berkow(ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway,N.J.; Thorn, et al. Harrison's Principles of Internal Medicine,McGraw-Hill, N.Y.; and Weatherall, et al. (eds.) Oxford Textbook ofMedicine, Oxford University Press, Oxford. Many other medical conditionsand diseases will be responsive to treatment by an agonist or antagonistprovided herein. See, e.g., Stites and Ten (eds.; 1991) Basic andClinical Immunology Appleton and Lange, Norwalk, Conn.; and Samter, etal. (eds.) Immunological Diseases Little, Brown and Co. Other likelyindications for treatment include bone remodeling, sexual dysfunction,prevention of neurodegenerative diseases, dementia, stress, and others.These problems should be susceptible to prevention or treatment usingcompositions provided herein.

Recombinant cytokine receptors, muteins, agonist or antagonistantibodies thereto, or antibodies can be purified and then administeredto a patient. These reagents can be combined for therapeutic use withadditional active ingredients, e.g., in conventional pharmaceuticallyacceptable carriers or diluents, along with physiologically innocuousstabilizers and excipients. These combinations can be sterile, e.g.,filtered, and placed into dosage forms as by lyophilization in dosagevials or storage in stabilized aqueous preparations. This invention alsocontemplates use of antibodies or binding fragments thereof which arenot complement binding.

Ligand screening using cytokine receptor or fragments thereof can beperformed to identify molecules having binding affinity to thereceptors. Subsequent biological assays can then be utilized todetermine if a putative ligand can provide competitive binding, whichcan block intrinsic stimulating activity. Receptor fragments can be usedas a blocker or antagonist in that it blocks the activity of ligand.Likewise, a compound having intrinsic stimulating activity can activatethe receptor and is thus an agonist in that it simulates the activity ofligand, e.g., inducing signaling. This invention further contemplatesthe therapeutic use of antibodies to cytokine receptors as antagonists.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, reagent physiological life, pharmacological life, physiologicalstate of the patient, and other medicants administered. Thus, treatmentdosages should be titrated to optimize safety and efficacy. Typically,dosages used in vitro may provide useful guidance in the amounts usefulfor in situ administration of these reagents. Animal testing ofeffective doses for treatment of particular disorders will providefurther predictive indication of human dosage. Various considerationsare described, e.g., in Gilman, et al. (eds. 1990) Goodman and Gilman's:The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; andRemington's Pharmaceutical Sciences, 17th ed. (1990), Mack PublishingCo., Easton, Pa.; each of which is hereby incorporated herein byreference. Methods for administration are discussed therein and below,e.g., for oral, intravenous, intraperitoneal, or intramuscularadministration, transdermal diffusion, and others. Pharmaceuticallyacceptable carriers will include water, saline, buffers, and othercompounds described, e.g., in the Merck Index, Merck & Co., Rahway, N.J.Because of the likely high affinity binding, or turnover numbers,between a putative ligand and its receptors, low dosages of thesereagents would be initially expected to be effective. And the signalingpathway suggests extremely low amounts of ligand may have effect. Thus,dosage ranges would ordinarily be expected to be in amounts lower than 1mM concentrations, typically less than about 10 μM concentrations,usually less than about 100 nM, preferably less than about 10 pM(picomolar), and most preferably less than about 1 fM (femtomolar), withan appropriate carrier. Slow release formulations, or slow releaseapparatus will often be utilized for continuous administration.

Cytokine receptors, fragments thereof, and antibodies or its fragments,antagonists, and agonists, may be administered directly to the host tobe treated or, depending on the size of the compounds, it may bedesirable to conjugate them to carrier proteins such as ovalbumin orserum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationscomprise at least one active ingredient, as defined above, together withone or more acceptable carriers thereof. Each carrier must be bothpharmaceutically and physiologically acceptable in the sense of beingcompatible with the other ingredients and not injurious to the patient.Formulations include those suitable for oral, rectal, nasal, orparenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by methods well knownin the art of pharmacy. See, e.g., Gilman, et al. (eds. 1990) Goodmanand Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed.,Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed.(1990), Mack Publishing Co., Easton, Pa.; Avis, et al. (eds. 1993)Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY;Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: TabletsDekker, NY; and Lieberman, et al. (eds. 1990) Pharmaceutical DosageForms: Disperse Systems Dekker, NY. The therapy of this invention may becombined with or used in association with other therapeutic agents,particularly agonists or antagonists of other cytokine receptor familymembers.

IX. Screening

Drug screening using DCRS5 or fragments thereof can be performed toidentify compounds having binding affinity to the receptor subunit,including isolation of associated components. Subsequent biologicalassays can then be utilized to determine if the compound has intrinsicstimulating activity and is therefore a blocker or antagonist in that itblocks the activity of the ligand.

Moreover, matching of the p40/IL-B30 ligand with the functional receptorof DCRS3 with IL-12Rβ1, allows screening for antagonists and agonistswith a positive signaling control. Small molecule or antibody screeningcan be done.

One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant DNA moleculesexpressing the DCRS5 in combination with another cytokine receptorsubunit, e.g., the IL-12Rβ1. The signaling is believed to use STAT4.Cells may be isolated which express a receptor in isolation from otherfunctional receptors. Such cells, either in viable or fixed form, can beused for standard antibody/antigen or ligand/receptor binding assays.See also, Parce, et al. (1989) Science 246:243-247; and Owicki, et al.(1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitivemethods to detect cellular responses. Competitive assays areparticularly useful, where the cells are contacted and incubated with alabeled receptor or antibody having known binding affinity to theligand, such as 125I-antibody, and a test sample whose binding affinityto the binding composition is being measured. The bound and free labeledbinding compositions are then separated to assess the degree of ligandbinding. The amount of test compound bound is inversely proportional tothe amount of labeled receptor binding to the known source. Manytechniques can be used to separate bound from free ligand to assess thedegree of ligand binding. This separation step could typically involve aprocedure such as adhesion to filters followed by washing, adhesion toplastic followed by washing, or centrifugation of the cell membranes.Viable cells could also be used to screen for the effects of drugs oncytokine mediated functions, e.g., STAT4 signaling and others. Somedetection methods allow for elimination of a separation step, e.g., aproximity sensitive detection system.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the inventionsto the specific embodiments.

EXAMPLES I. General Methods

Some of the standard methods are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3, CSHPress, NY; or Ausubel, et al. (1987 and Supplements) Current Protocolsin Molecular Biology, Greene/Wiley, New York. Methods for proteinpurification include such methods as ammonium sulfate precipitation,column chromatography, electrophoresis, centrifugation, crystallization,and others. See, e.g., Ausubel, et al. (1987 and periodic supplements);Coligan, et al. (ed. 1996) and periodic supplements, Current ProtocolsIn Protein Science Greene/Wiley, New York; Deutscher (1990) “Guide toProtein Purification” in Methods in Enzymology, vol. 182, and othervolumes in this series; and manufacturer's literature on use of proteinpurification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad,Richmond, Calif. Combination with recombinant techniques allow fusion toappropriate segments, e.g., to a FLAG sequence or an equivalent whichcan be fused via a protease-removable sequence. See, e.g., Hochuli(1990) “Purification of Recombinant Proteins with Metal ChelateAbsorbent” in Setlow (ed.) Genetic Engineering, Principle and Methods12:87-98, Plenum Press, N.Y.; and Crowe, et al. (1992) QIAexpress: TheHigh Level Expression & Protein Purification System QUIAGEN, Inc.,Chatsworth, Calif.

Computer sequence analysis is performed, e.g., using available softwareprograms, including those from the GCG (U. Wisconsin) and GenBanksources. Public sequence databases were also used, e.g., from GenBankand others.

Many techniques applicable to IL-10 receptors may be applied to theDCRS5, as described, e.g., in U.S. Pat. No. 5,789,192 (IL-10 receptor),which is incorporated herein by reference.

II. Functional Cloning

It was observed that anti-hIL-12Rβ1 antibody blocked responses of humanT cells to p40/IL-B30, and the p40/IL-B30 bound to IL-12Rβ1. Thissuggested that IL-12Rβ1 was one subunit of the receptor complex forp40/IL-B30.

A mouse T cell population was identified which responded to p40/IL-B30but not to IL-12, and another population which responded to IL-12 butnot p40/IL-B30. In addition, it was observed that Ba/F3 cells expressingrecombinant mIL-12Rβ1 and mIL-12Rβ2 responded to IL-12, but not top40/IL-B30. These results collectively indicated that the receptorcomplex for p40/IL-B30 contained the IL-12Rβ1 and at least one othersubunit which was not IL-12Rβ2. Accordingly an expression cloningstrategy was devised to isolate this second receptor component.

A cDNA library was prepared from mRNA isolated from Kit225 cells, anIL-2-dependent human T cell line which responds to both IL-12 andp40/IL-B30. The cDNA library was made using a retroviral expressionvector, pMX. Ba/F3 cells expressing recombinant hIL-12Rβ1 were infectedwith this cDNA library, allowed to recover for 3-4 days in IL-3, thenwashed and plated at ˜15,000 cells/well in 96 well plates in mediumcontaining 50 ng/ml hyper-hp40/hIL-B30. See WO 01/18051. Cultures weresupplemented every ˜5 days with additional hyper-hp40/hIL-B30. Afterapproximately two weeks 5-10% of the wells exhibited cell growth. Cellswere recovered from each well, expanded individually in larger culturesin hyper-hp40/hIL-B30, and tested for growth dependence onhyper-hp40/hIL-B30.

Cells which were p40/IL-B30-dependent for growth were analyzed by PCRfor retroviral cDNA inserts. Out of more than 40 isolates analyzed, allbut one contained cDNAs encoding the novel receptor DCRS5. Thiscandidate human cDNA was cloned in an expression vector and transfectedinto Ba/F3 cells expressing hIL-12Rβ1. These cells became responsive top40/IL-B30; thus we concluded that the novel cDNA encoded the desiredDCRS5, functionally an IL-B30 receptor subunit.

III. Features of Full-Length DCRS5; Chromosomal Localization

The cytoplasmic domain of DCRS5 is not overall closely related to othercytokine receptor cytoplasmic domains, a common observation in thisfamily of molecules. The cytoplasmic domain contains seven tyr residues,at least three of which are part of recognizable SH2-binding motifs:YEDI, YKPQ, and YFPQ. The YEDI motif is similar to identified bindingsites for the tyrosine phosphatase shp2. The latter two motifs are verysimilar to sequences known to bind Stat1/Stat3, or Stat3, respectively.The YKPQ motif, together with nearby flanking sequences, also resemblesto a degree the motifs in Stat4 and IL-12Rβ2 which are known to bindStat1-3. This is consistent with preliminary data suggesting thatp40/IL-B30, like IL-12, activates Stat4.

PCR primers derived from the DCRS5 sequence are used to probe a humancDNA library. Sequences may be derived, e.g., from Table 1, preferablythose adjacent the ends of sequences. Full length cDNAs for primate,rodent, or other species DCRS5 are cloned, e.g., by DNA hybridizationscreening of λgt10 phage. PCR reactions are conducted using T. aquaticusTaqplus DNA polymerase (Stratagene) under appropriate conditions.

Chromosome spreads are prepared. In situ hybridization is performed onchromosome preparations obtained from phytohemagglutinin-stimulatedhuman lymphocytes cultured for 72 h. 5-bromodeoxyuridine was added forthe final seven hours of culture (60 μg/ml of medium), to ensure aposthybridization chromosomal banding of good quality.

A PCR fragment, amplified with the help of primers, is cloned into anappropriate vector. The vector is labeled by nick-translation with ³H.The radiolabeled probe is hybridized to metaphase spreads at finalconcentration of 200 ng/ml of hybridization solution as described inMattei, et al. (1985) Hum. Genet. 69:327-331.

After coating with nuclear track emulsion (KODAK NTB2), slides areexposed. To avoid any slipping of silver grains during the bandingprocedure, chromosome spreads are first stained with buffered Giemsasolution and metaphase photographed. R-banding is then performed by thefluorochrome-photolysis-Giemsa (FPG) method and metaphasesrephotographed before analysis.

Similar appropriate methods are used for other species.

IV. Localization of DCRS5 mRNA

Human multiple tissue (Cat# 1,2) and cancer cell line blots (Cat#7757-1), containing approximately 2 μg of poly (A) ⁺RNA per lane, arepurchased from Clontech (Palo Alto, Calif.). Probes are radiolabeledwith [α-³²P] dATP, e.g., using the Amersham Rediprime random primerlabeling kit (RPN1633). Prehybridization and hybridizations areperformed, e.g., at 65° C. in 0.5 M Na₂HPO₄, 7% SDS, 0.5 M EDTA (pH8.0). High stringency washes are conducted, e.g., at 65° C. with twoinitial washes in 2×SSC, 0.1% SDS for 40 min followed by a subsequentwash in 0.1×SSC, 0.1% SDS for 20 min. Membranes are then exposed at −70°C. to X-Ray film (Kodak) in the presence of intensifying screens. Moredetailed studies by cDNA library Southerns are performed with selectedappropriate human DCRS5 clones to examine their expression inhemopoietic or other cell subsets.

Alternatively, two appropriate primers are selected from Table 1. RT-PCRis used on an appropriate mRNA sample selected for the presence ofmessage to produce a cDNA, e.g., a sample which expresses the gene.

Full length clones may be isolated by hybridization of cDNA librariesfrom appropriate tissues pre-selected by PCR signal. Northern blots canbe performed.

Message for genes encoding DCRS5 will be assayed by appropriatetechnology, e.g., PCR, immunoassay, hybridization, or otherwise. Tissueand organ cDNA preparations are available, e.g., from Clontech, MountainView, Calif. Identification of sources of natural expression are useful,as described. And the identification of the functional receptor subunitpairing allows for prediction of what cells express the combination ofreceptor subunits which will result in a physiological responsiveness toeach of the cytokine ligands.

For mouse distribution, e.g., Southern Analysis can be performed: DNA (5μg) from a primary amplified cDNA library was digested with appropriaterestriction enzymes to release the inserts, run on a 1% agarose gel andtransferred to a nylon membrane (Schleicher and Schuell, Keene, N.Y.).

Samples for mouse mRNA isolation may include: resting mouse fibroblasticL cell line (C200); Braf:ER (Braf fusion to estrogen receptor)transfected cells, control (C201); T cells, TH1 polarized (Mell4 bright,CD4+ cells from spleen, polarized for 7 days with IFN-γ and anti IL-4;T200); T cells, TH2 polarized (Mell4 bright, CD4+ cells from spleen,polarized for 7 days with IL-4 and anti-IFN-γ; T201); T cells, highlyTH1 polarized (see Openshaw, et al. (1995) J. Exp. Med. 182:1357-1367;activated with anti-CD3 for 2, 6, 16 h pooled; T202); T cells, highlyTH2 polarized (see Openshaw, et al. (1995) J. Exp. Med. 182:1357-1367;activated with anti-CD3 for 2, 6, 16 h pooled; T203); CD44-CD25+ pre Tcells, sorted from thymus (T204); TH1 T cell clone D1.1, resting for 3weeks after last stimulation with antigen (T205); TH1 T cell clone D1.1,10 μg/ml ConA stimulated 15 h (T206); TH2 T cell clone CDC35, restingfor 3 weeks after last stimulation with antigen (T207); TH2 T cell cloneCDC35, 10 μg/ml ConA stimulated 15 h (T208); Mell4+ naïve T cells fromspleen, resting (T209); Mell4+T cells, polarized to Th1 withIFN-γ/IL-12/anti-IL-4 for 6, 12, 24 h pooled (T210); Mell4+T cells,polarized to Th2 with IL-4/anti-IFN-γ for 6, 13, 24 h pooled (T211);unstimulated mature B cell leukemia cell line A20 (B200); unstimulated Bcell line CH12 (B201); unstimulated large B cells from spleen (B202); Bcells from total spleen, LPS activated (B203); metrizamide enricheddendritic cells from spleen, resting (D200); dendritic cells from bonemarrow, resting (D201); monocyte cell line RAW 264.7 activated with LPS4 h (M200); bone-marrow macrophages derived with GM and M-CSF (M201);macrophage cell line J774, resting (M202); macrophage cell lineJ774+LPS+anti-IL-10 at 0.5, 1, 3, 6, 12 h pooled (M203); macrophage cellline J774+LPS+IL-10 at 0.5, 1, 3, 5, 12 h pooled (M204); aerosolchallenged mouse lung tissue, Th2 primers, aerosol OVA challenge 7, 14,23 h pooled (see Garlisi, et al. (1995) Clinical Immunology andImmunopathology 75:75-83; X206); Nippostrongulus-infected lung tissue(see Coffman, et al. (1989) Science 245:308-310; X200); total adultlung, normal (O200); total lung, rag-1 (see Schwarz, et al. (1993)Immunodeficiency 4:249-252; 0205); IL-10 K.O. spleen (see Kuhn, et al.(1991) Cell 75:263-274; X201); total adult spleen, normal (O201); totalspleen, rag-1 (O207); IL-10 K.O. Peyer's patches (O202); total Peyer'spatches, normal (O210); IL-10 K.O. mesenteric lymph nodes (X203); totalmesenteric lymph nodes, normal (O211); IL-10 K.O. colon (X203); totalcolon, normal (O212); NOD mouse pancreas (see Makino, et al. (1980)Jikken Dobutsu 29:1-13; X205); total thymus, rag-1 (O208); total kidney,rag-1 (O209); total heart, rag-1 (O202); total brain, rag-1 (O203);total testes, rag-1 (O204); total liver, rag-1 (O206); rat normal jointtissue (O300); and rat arthritic joint tissue (X300).

Samples for human mRNA isolation may include: peripheral bloodmononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells),resting (T100); peripheral blood mononuclear cells, activated withanti-CD3 for 2, 6, 12 h pooled (T101); T cell, TH0 clone Mot 72, resting(T102); T cell, TH0 clone Mot 72, activated with anti-CD28 and anti-CD3for 3, 6, 12 h pooled (T103); T cell, TH0 clone Mot 72, anergic treatedwith specific peptide for 2, 7, 12 h pooled (T104); T cell, TH1 cloneHY06, resting (T107); T cell, TH1 clone HY06, activated with anti-CD28and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1 clone HY06,anergic treated with specific peptide for 2, 6, 12 h pooled (T109); Tcell, TH2 clone HY935, resting (T110); T cell, TH2 clone HY935,activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T111); Tcells CD4+CD45RO− T cells polarized 27 days in anti-CD28, IL-4, and antiIFN-γ, TH2 polarized, activated with anti-CD3 and anti-CD28 4 h (T116);T cell tumor lines Jurkat and Hut78, resting (T117); T cell clones,pooled AD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting (T118);T cell random γδ T cell clones, resting (T119); Splenocytes, resting(B100); Splenocytes, activated with anti-CD40 and IL-4 (B101); B cellEBV lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting (B102);B cell line JY, activated with PMA and ionomycin for 1, 6 h pooled(B103); NK 20 clones pooled, resting (K100); NK 20 clones pooled,activated with PMA and ionomycin for 6 h (K101); NKL clone, derived fromperipheral blood of LGL leukemia patient, IL-2 treated (K106); NKcytotoxic clone 640-A30-1, resting (K107); hematopoietic precursor lineTF1, activated with PMA and ionomycin for 1, 6 h pooled (C100); U937premonocytic line, resting (M100); U937 premonocytic line, activatedwith PMA and ionomycin for 1, 6 h pooled (M101); elutriated monocytes,activated with LPS, IFNγ, anti-IL-10 for 1, 2, 6, 12, 24 h pooled(M102); elutriated monocytes, activated with LPS, IFNγ, IL-10 for 1, 2,6, 12, 24 h pooled (M103); elutriated monocytes, activated with LPS,IFNγ, anti-IL-10 for 4, 16 h pooled (M106); elutriated monocytes,activated with LPS, IFNγ, IL-10 for 4, 16 h pooled (M107); elutriatedmonocytes, activated LPS for 1 h (M108); elutriated monocytes, activatedLPS for 6 h (M109); DC 70% CD1a+, from CD34+GM-CSF, TNFα 12 days,resting (D101); DC 70% CD1a+, from CD34+GM-CSF, TNFα 12 days, activatedwith PMA and ionomycin for 1 hr (D102); DC 70% CD1a+, from CD34+GM-CSF,TNFα 12 days, activated with PMA and ionomycin for 6 hr (D103); DC 95%CD1a+, from CD34+GM-CSF, TNFα 12 days FACS sorted, activated with PMAand ionomycin for 1, 6 hr pooled (D104); DC 95% CD14+, ex CD34+GM-CSF,TNFα 12 days FACS sorted, activated with PMA and ionomycin 1, 6 hrpooled (D105); DC CD1a+CD86+, from CD34+GM-CSF, TNFα 12 days FAC sorted,activated with PMA and ionomycin for 1, 6 h pooled (K106); DC frommonocytes GM-CSF, IL-4 5 days, resting (D107); DC from monocytes GM-CSF,IL-4 5 days, resting (D108); DC from monocytes GM-CSF, IL-4 5 days,activated LPS 4, 16 h pooled (D109); DC from monocytes GM-CSF, IL-4 5days, activated TNFα, monocyte supe for 4, 16 h pooled (D110); leiomyomaL11 benign tumor (X101); normal myometrium M5 (O115); malignantleiomyosarcoma GS1 (X103); lung fibroblast sarcoma line MRCS, activatedwith PMA and ionomycin for 1, 6 h pooled (C101); kidney epithelialcarcinoma cell line CHA, activated with PMA and ionomycin for 1, 6 hpooled (C102); kidney fetal 28 wk male (O100); lung fetal 28 wk male(O101); liver fetal 28 wk male (O102); heart fetal 28 wk male (O103);brain fetal 28 wk male (O104); gallbladder fetal 28 wk male (O106);small intestine fetal 28 wk male (O107); adipose tissue fetal 28 wk male(O108); ovary fetal 25 wk female (O109); uterus fetal 25 wk female(O110); testes fetal 28 wk male (O111); spleen fetal 28 wk male (O112);adult placenta 28 wk (O113); and tonsil inflamed, from 12 year old(X100).

Similar samples may isolated in other species for evaluation.

V. Cloning of Species Counterparts of DCRS5

Various strategies are used to obtain species counterparts of the DCRS5,preferably from other primates or rodents. One method is by crosshybridization using closely related species DNA probes. It may be usefulto go into evolutionarily similar species as intermediate steps. Anothermethod is by using specific PCR primers based on the identification ofblocks of similarity or difference between genes, e.g., areas of highlyconserved or nonconserved polypeptide or nucleotide sequence.

Database searches may identify similar sequences and allow production ofappropriate probes.

VI. Production of Mammalian DCRS5 Protein

An appropriate, e.g., GST, fusion construct is engineered forexpression, e.g., in E. coli. For example, a mouse IGIF pGex plasmid isconstructed and transformed into E. coli. Freshly transformed cells aregrown, e.g., in LB medium containing 50 μg/ml ampicillin and inducedwith IPTG (Sigma, St. Louis, Mo.). After overnight induction, thebacteria are harvested and the pellets containing the DCRS5 protein areisolated. The pellets are homogenized, e.g., in TE buffer (50 mMTris-base pH 8.0, 10 mM EDTA and 2 mM pefabloc) in 2 liters. Thismaterial is passed through a microfluidizer (Microfluidics, Newton,Mass.) three times. The fluidized supernatant is spun down on a SorvallGS-3 rotor for 1 h at 13,000 rpm. The resulting supernatant containingthe cytokine receptor protein is filtered and passed over aglutathione-SEPHAROSE column equilibrated in 50 mM Tris-base pH 8.0. Thefractions containing the DCRS5-GST fusion protein are pooled andcleaved, e.g., with thrombin (Enzyme Research Laboratories, Inc., SouthBend, Ind.). The cleaved pool is then passed over a Q-SEPHAROSE columnequilibrated in 50 mM Tris-base. Fractions containing DCRS5 are pooledand diluted in cold distilled H₂O, to lower the conductivity, and passedback over a fresh Q-Sepharose column, alone or in succession with animmunoaffinity antibody column. Fractions containing the DCRS5 proteinare pooled, aliquoted, and stored in the −70 bC freezer.

Comparison of the CD spectrum with cytokine receptor protein may suggestthat the protein is correctly folded. See Hazuda, et al. (1969) J. Biol.Chem. 264:1689-1693.

VII. Preparation of Antibodies Specific for DCRS5

Inbred Balb/c mice are immunized intraperitoneally with recombinantforms of the protein, e.g., purified DCRS5 or stable transfected NIH-3T3cells. Animals are boosted at appropriate time points with protein, withor without additional adjuvant, to further stimulate antibodyproduction. Serum is collected, or hybridomas produced with harvestedspleens.

Alternatively, Balb/c mice are immunized with cells transformed with thegene or fragments thereof, either endogenous or exogenous cells, or withisolated membranes enriched for expression of the antigen. Serum iscollected at the appropriate time, typically after numerous furtheradministrations. Various gene therapy techniques may be useful, e.g., inproducing protein in situ, for generating an immune response. Serum orantibody preparations may be cross-absorbed or immunoselected to preparesubstantially purified antibodies of defined specificity and highaffinity.

Monoclonal antibodies may be made. For example, splenocytes are fusedwith an appropriate fusion partner and hybridomas are selected in growthmedium by standard procedures. Hybridoma supernatants are screened forthe presence of antibodies which bind to the DCRS5, e.g., by ELISA orother assay. Antibodies which specifically recognize specific DCRS5embodiments may also be selected or prepared.

In another method, synthetic peptides or purified protein are presentedto an immune system to generate monoclonal or polyclonal antibodies.See, e.g., Coligan (ed. 1991) Current Protocols in ImmunologyWiley/Greene; and Harlow and Lane (1989) Antibodies: A Laboratory ManualCold Spring Harbor Press. In appropriate situations, the binding reagentis either labeled as described above, e.g., fluorescence or otherwise,or immobilized to a substrate for panning methods. Nucleic acids mayalso be introduced into cells in an animal to produce the antigen, whichserves to elicit an immune response. See, e.g., Wang, et al. (1993)Proc. Nat'l. Acad. Sci. 90:4156-4160; Barry, et al. (1994) BioTechniques16:616-619; and Xiang, et al. (1995) Immunity 2: 129-135.

VIII. Production of Fusion Proteins with DCRS5

Various fusion constructs are made with DCRS5, including embodimentscombining such with IL-12Rβ1 sequence. A portion of the appropriate geneis fused to an epitope tag, e.g., a FLAG tag, or to a two hybrid systemconstruct. See, e.g., Fields and Song (1989) Nature 340:245-246.

The epitope tag may be used in an expression cloning procedure withdetection with anti-FLAG antibodies to detect a binding partner, e.g.,ligand for the respective cytokine receptor. The two hybrid system mayalso be used to isolate proteins which specifically bind to DCRS5.

IX. Structure Activity Relationship

Information on the criticality of particular residues is determinedusing standard procedures and analysis. Standard mutagenesis analysis isperformed, e.g., by generating many different variants at determinedpositions, e.g., at the positions identified above, and evaluatingbiological activities of the variants. This may be performed to theextent of determining positions which modify activity, or to focus onspecific positions to determine the residues which can be substituted toeither retain, block, or modulate biological activity.

Alternatively, analysis of natural variants can indicate what positionstolerate natural mutations. This may result from populational analysisof variation among individuals, or across strains or species. Samplesfrom selected individuals are analyzed, e.g., by PCR analysis andsequencing. This allows evaluation of population polymorphisms.

X. Coexpression of DCRS5 and IL-12Rb1

A vector, or vectors, encoding the respective genes may be transfectedinto a cell.

Preferably, such vector will have selection markers to identify whichcells have successfully been transformed. Coexpression of the two geneswill allow the gene products to properly associate to form activereceptor complexes.

Alternatively, use of methods causing association of functional dimersare available. See, e.g., O'Shea, et al. (1989) Science 245:646-648;Kostelny, et al. (1992) J. Immunol. 148:1547-1553; and Patel, et al.(1996) J. Biol. Chem. 271:30386-30391. Expression of extracellulardomains, and physical association, e.g., driven by Fos/Jun leucinezipper affinity, will result in ligand binding constructs which shouldact as binding compounds for diagnostic or therapeutic uses.

All citations herein are incorporated herein by reference to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled; and the invention is notto be limited by the specific embodiments that have been presentedherein by way of example.

1. A substantially pure or recombinant polypeptide comprising at leastten contiguous amino acids of the intracellular portion of SEQ ID NO: 2.2. The polypeptide of claim 1, wherein: a) said polypeptide comprises atleast 25 contiguous amino acids of the intracellular portion of SEQ IDNO: 2; b) said polypeptide is recombinant, comprising the intracellularportion of SEQ ID NO: 2; c) said polypeptide further comprises at leastten contiguous amino acids of the non-intracellular portion of SEQ IDNO: 2; d) said polypeptide comprises at least 25 amino acids of theextracellular portion of SEQ ID NO: 2; e) said polypeptide comprises themature SEQ ID NO: 2; or f) said polypeptide is a substantially purenatural polypeptide.
 3. The recombinant polypeptide of claim 1, which:a) consists of the mature sequence of Table 1; b) is an unglycosylatedpolypeptide; c) is from a human; d) comprises at least 40 contiguousamino acids of SEQ ID NO: 2; e) exhibits at least three nonoverlappingsegments of at least fifteen contiguous amino acids of SEQ ID NO: 2; f)is a natural polymorphic variant of SEQ ID NO: 2; g) has a length atleast about 30 amino acids; h) exhibits at least two non-overlappingepitopes which are specific for a primate DCRS5; i) has a molecularweight of at least 30 kD with natural glycosylation; j) is a syntheticpolypeptide; k) is in a sterile form; l) is in an aqueous or bufferedsolution; m) is attached to a solid substrate; n) is conjugated toanother chemical moiety; or o) is physically associated with an IL-12Rβ1polypeptide;
 4. A composition of matter selected from: a) asubstantially pure or recombinant polypeptide comprising at least twodistinct nonoverlapping segments of at least six contiguous amino acidsof the intracellular portion of SEQ ID NO: 2 b.) a substantially pure orrecombinant polypeptide comprising at least 12 contiguous amino acids ofthe intracellular portion of SEQ ID NO:2; or c) a substantially purenatural sequence polypeptide comprising mature SEQ ID NO:
 2. 5. Thepolypeptide: 1) of claim 4a, wherein: a) said distinct nonoverlappingsegments: i) include one of at least twelve amino acids; ii) include oneof at least seven amino acids and a second of at least nine amino acids;iii) include a third distinct segment of at least six amino acids; oriv) comprise one of R355-L373, P378-L405, V407-D426, K428-D439,P441-V452, I454-G460, I465-T587, or N592-606; or b) said polypeptidefurther comprises at least two distinct nonoverlapping segments of atleast six contiguous amino acids of the extracellular portion of SEQ IDNO: 2; 2) of claim 4b, wherein: a) said at least twelve contiguous aminoacid segment comprises one of R355-L373, P378-L405, V407-D426,K428-D439, P441-V452, I454-G460, I465-T587, or N592-606; or b) saidpolypeptide further comprises at least two distinct nonoverlappingsegments of at least six contiguous amino acids of the extracellularportion of SEQ ID NO: 2; or 3) of claim 4c, further comprising apurification or detection epitope.
 6. The polypeptide of claim 4, which:a) consists of the mature sequence of Table 1; b) is an unglycosylatedpolypeptide; c) is from a human; d) comprises at least 40 contiguousamino acids of SEQ ID NO: 2; e) exhibits at least three nonoverlappingsegments of at least fifteen contiguous amino acids of SEQ ID NO: 2; f)is a natural polymorphic variant of SEQ ID NO: 2; g) has a length atleast about 30 amino acids; h) exhibits at least two non-overlappingepitopes which are specific for a primate DCRS5; i) has a molecularweight of at least 30 kD with natural glycosylation; j) is a syntheticpolypeptide; k) is in a sterile form; l) is in an aqueous or bufferedsolution; m) is attached to a solid substrate; n) is conjugated toanother chemical moiety; or o) is physically associated with an IL-12Rβ1polypeptide.
 7. A composition comprising: a) a substantially purepolypeptide of claim 4 combined with the IL-12Rβ1 protein; or b) saidpolypeptide of claim 4 and a carrier, wherein said carrier is: i) anaqueous compound, including water, saline, and/or buffer; and/or ii)formulated for oral, rectal, nasal, topical, or parenteraladministration.
 8. A kit comprising a polypeptide of claim 4, and: a) acompartment comprising said polypeptide; b) a compartment comprising anIL-12Rβ1 polypeptide; c) a compartment comprising a p40, IL-B30, orp40/IL-B30 polypeptide; or d) instructions for use or disposal ofreagents in said kit.
 9. A binding compound comprising an antigenbinding site from an antibody, which specifically binds to saidintracellular portion of said polypeptide of claim 1, wherein: a) saidbinding compound is in a container; b) said polypeptide is from a human;c) said binding compound is an Fv, Fab, or Fab2 fragment; d) saidbinding compound is conjugated to another chemical moiety; or e) saidantibody: i) is raised against a peptide sequence of a maturepolypeptide of Table 1; ii) is raised against a mature DCRS5; iii) israised to a purified human DCRS5; iv) is immunoselected; v) is apolyclonal antibody; luorescent label. vi) binds to a denatured DCRS5;vii) exhibits a Kd to antigen of at least 30 μM; viii) is attached to asolid substrate, including a bead or plastic membrane; ix) is in asterile composition; or x) is detectably labeled, including aradioactive or fluorescent label.
 10. A kit comprising said bindingcompound of claim 9, and: a) a compartment comprising said bindingcompound; b) a compartment comprising: i) a p40 polypeptide; ii) anIL-B30 polypeptide; iii) a DCRS5 polypeptide; and/or iv) an IL-12Rβ1polypeptide; c) a compartment comprising an antibody which bindsselectively to: i) a p40 polypeptide; ii) an IL-B30 polypeptide; iii) aDCRS5 polypeptide; and/or iv) an IL-12Rβ1 polypeptide; or d)instructions for use or disposal of reagents in said kit.
 11. A methodof producing an antigen:antibody complex, comprising contacting underappropriate conditions a primate DCRS5 polypeptide with an antibody ofclaim 9, thereby allowing said complex to form.
 12. The method of claim11, wherein: a) said complex is purified from other cytokine receptors;b) said complex is purified from other antibody; c) said contacting iswith a sample comprising an interferon; d) said contacting allowsquantitative detection of said antigen; e) said contacting is with asample comprising said antibody; or f) said contacting allowsquantitative detection of said antibody.
 13. A composition comprising:a) a sterile binding compound of claim 9, or b) said binding compound ofclaim 9 and a carrier, wherein said carrier is: i) an aqueous compound,including water, saline, and/or buffer; and/or ii) formulated for oral,rectal, nasal, topical, or parenteral administration.
 14. An isolated orrecombinant nucleic acid encoding said DCRS5 polypeptide of claim 1,wherein said: a) DCRS5 is from a human; or b) said nucleic acid: i)encodes an antigenic peptide sequence of Table 1; ii) encodes aplurality of antigenic peptide sequences of Table 1; iii) exhibitsidentity over at least thirteen nucleotides to a natural cDNA encodingsaid segment; iv) is an expression vector; v) further comprises anorigin of replication; vi) is from a natural source; vii) comprises adetectable label; viii) comprises synthetic nucleotide sequence; ix) isless than 6 kb, preferably less than 3 kb; x) is from a primate; xi)comprises a natural full length coding sequence; xii) is a hybridizationprobe for a gene encoding said DCRS5; or xiii) is a PCR primer, PCRproduct, or mutagenesis primer.
 15. A cell or tissue comprising saidrecombinant nucleic acid of claim
 14. 16. The cell of claim 15, whereinsaid cell is: a) a prokaryotic cell; b) a eukaryotic cell; c) abacterial cell; d) a yeast cell; e) an insect cell; f) a mammalian cell;g) a mouse cell; h) a primate cell; or i) a human cell.
 17. A kitcomprising said nucleic acid of claim 14, and: a) a compartmentcomprising said nucleic acid; b) a compartment comprising a nucleic acidencoding: i) a p40 polypeptide; ii) an IL-B30 polypeptide; iii) a DCRS5polypeptide; and/or iv) an IL-12Rβ1 polypeptide; c) a compartmentcomprising: i) a p40 polypeptide; ii) an IL-B30 polypeptide; iii) aDCRS5 polypeptide; and/or iv) an IL-12Rβ1 polypeptide; d) a compartmentcomprising an antibody which selectively binds to: i) a p40 polypeptide;ii) an IL-B30 polypeptide; iii) a DCRS5 polypeptide; and/or iv) anIL-12Rβ1 polypeptide; or e) instructions for use or disposal of reagentsin said kit.
 18. A nucleic acid which: a) hybridizes under washconditions of 30 minutes at 30° C. and less than 2M salt to the portionof SEQ ID NO: 1 encoding the intracellular portion; or b) exhibitsidentity over a stretch of at least about 30 nucleotides to theintracellular portion of a primate DCRS5.
 19. The nucleic acid of claim18, wherein: a) said wash conditions are at 45° C. and/or 500 mM salt;or b) said stretch is at least 55 nucleotides.
 20. The nucleic acid ofclaim 18, wherein: a) said wash conditions are at 55° C. and/or 150 mMsalt; or b) said stretch is at least 75 nucleotides.
 21. A method ofmodulating physiology or development of a cell comprising contactingsaid cell with: a) an antagonist of p40/IL-B30 which is a complexcomprising: i) the extracellular portion of a primate DCRS5; and/or ii)the extracellular portion of a primate IL-12R b) an antagonist ofp40/IL-B30 which is an antibody which binds a complex comprising: i)primate DCRS5; and/or ii) primate IL-12Rβ1; c) an antagonist ofp40/IL-B30 which is an antibody which bonds to DCRS5; d) an antagonistof p40/IL-B30 which is an antibody to IL-12Rβ1; e) an antagonist ofp40/IL-B30 which is an antisense nucleic acid to DCRS5 or IL-12Rβ1; orf) an agonist of p40/IL-B30 which is an antibody which binds a complexcomprising: i) primate DCRS5; and/or ii) primate IL-12Rβ1.
 22. Themethod of claim 21, wherein said contacting is with an antagonist, and:a) said contacting is in combination with an antagonist to: i) IL-12;ii) IL-18; iii) TNF; or iv) IFNγ; or b) said cell is from a host which:i) exhibits signs or symptoms of a chronic Th1 mediated disease; ii)exhibits symptoms or signs of multiple sclerosis, rheumatoid arthritis,osteoarthritis, inflammatory bowel disease, diabetes, psoriasis, orsepsis; or iii) receives an allogeneic transplant.
 23. The method ofclaim 21, wherein said contacting is with an agonist, and: a) saidcontacting is in combination with: i) IL-12; ii) IL-18; iii) TNF; or iv)IFN b) said cell is from a host which: i) exhibits signs or symptoms ofa chronic TH2 response; ii) suffers from a tumor, viral, or fungalgrowth; iii) receives a vaccine; or iv) suffers from an allergicresponse.